Methods and compositions for treating cancers using antisense

ABSTRACT

The present disclosure relates to compositions and methods for treating cancers using antisense (AS) nucleic acids directed against Insulin-like Growth Factor 1 Receptor (IGF-1R). The AS may be administered to the patients systemically, or may be used to produce an autologous cancer cell vaccine. In embodiments, the AS are provided in an implantable irradiated biodiffusion chamber comprising tumor cells and an effective amount of the AS. The chambers are irradiated and implanted in the abdomen of subjects and stimulate an immune response that attacks tumors distally. The compositions and methods disclosed herein may be used to treat many different kinds of cancer, for example glioblastoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/469,003 filed on Mar. 9, 2017, and 62/629,972, filed on Feb. 13,2018, each entitled “Methods and Compositions for Treating Cancers UsingAntisense,” the disclosure of each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for treatingcancers using antisense nucleic acids directed against Insulin-likeGrowth Factor-1 Receptor (IGF-1R). The present disclosure also relatesto compositions and methods for treating cancers by treating subjectswith at least one implantable irradiated biodiffusion chamber (see U.S.Pat. No. 6,541,036 and PCT/US2016/026970, which are incorporated hereinby reference in their entireties) comprising tumor cells and anantisense nucleic acid directed against IGF-1R.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated by reference in their entirety: a computer readable formatcopy of the Sequence Listing (filename: IMVX_005_02US_SeqList.txt, daterecorded Mar. 8, 2018, file size 12 kilobytes).

BACKGROUND

Despite advances in cancer therapy, the prognosis for malignant glioma,particularly glioblastoma multiforme, and many other cancers remainspoor. Modifications of standard treatments such as, for example,chemotherapy, external beam radiation, and brachytherapy provide onlysmall increments of improvement in both progression-free survival andoverall survival. Immunotherapy trials, although promising in theory,have not addressed the challenges created by solid tumors. For thetreatment of glioma, the National Cancer Institute estimates an annualincidence of around 28,000 cases annually which increases to over 50,000if patients with recurrent gliomas are included. Therefore, there is aneed in the art to obtain new and improved treatments for cancers, andcancers of the brain in particular.

SUMMARY OF THE INVENTION

The present disclosure demonstrates that an antisenseoligodeoxynucleotide (AS-ODN) targeting the insulin-like growth factorreceptor-1 (IGF-1R) effectively stimulates a response in a subject thattreats cancer when used in the therapeutic approaches described herein.In particular aspects, methods are effective for treating cancer in apatient as part of an autologous cancer cell vaccine alone or,optionally, along with systemic administration. In preferred approaches,the methods disclosed herein provide effective cancer therapy as amonotherapy; i.e. in the absence of chemotherapy and in the absence ofradiation therapy.

In embodiments, the present disclosure provides a biodiffusion chamberfor implantation into a subject suffering from a tumor, the biodiffusionchamber comprising irradiated tumor cells and irradiated insulin-likegrowth factor receptor-1 antisense oligodeoxynucleotide (IGF-1R AS ODN).In embodiments, the tumor cells are removed from a resection site of thesubject.

In embodiments, the present disclosure provides a diffusion chambercomprising irradiated IGF-1R AS ODN and irradiated, adhesion-enriched,morselized tumor cells; wherein the biodiffusion chamber comprises amembrane that is impermeable to the cells and permeable to the IGF-1R ASODN.

In embodiments, the tumor cells are removed from the resection siteusing an endoscopic device. In further embodiments, the tumor cells areremoved from the resection site using a tissue morselator. In otherembodiments, the tissue morselator comprises a high-speed reciprocatinginner cannula within a stationary outer cannula. The outer cannula maycomprise a side aperture, and further wherein the tumor cells are drawninto the side aperture by electronically controlled variable suction. Inembodiments, the tissue morselator does not produce heat at theresection site. In still further embodiments, the tumor cells areenriched for nestin expression before they are placed into thebiodiffusion chamber. In some embodiments, implantation of the chamberinhibits regrowth of the tumor in the subject. In some embodiments,implantation of the chamber inhibits regrowth of the tumor for at least3 months, at least 6 months, at least 12 months, or at least 36 months.

In additional embodiments, the present disclosure provides a method forpreparing a biodiffusion chamber for implantation into a subjectsuffering from a tumor, the method comprising placing tumor cells intothe biodiffusion chamber in the presence of an IGF-1R AS ODN, andirradiating the biodiffusion chamber, wherein the tumor cells areremoved from a resection site in the subject using a tissue morselatorthat does not produce heat at the resection site. Typically, multiplechambers are used. For example, about 10 chambers, or about 20 chambers.Advantageously, an optimal anti-tumor response is obtained when thenumber of cells in the chamber is about 750,000 to about 1,250,000; forexample about 1,000,000 per chamber where 20 chambers are implanted.

In some embodiments, the tissue morselator is an endoscopic device. Infurther embodiments, the tissue morselator comprises a high-speedreciprocating inner cannula within a stationary outer cannula. Inadditional embodiments, the outer cannula comprises a side aperture, andthe tumor cells are drawn into the side aperture by electronicallycontrolled variable suction.

In embodiments, the present disclosure provides a method of treating asubject suffering from a tumor, the method comprising implanting one ormore biodiffusion chambers into the subject, wherein the one or morebiodiffusion chambers comprise irradiated tumor cells, and irradiatedinsulin-like growth factor receptor-1 antisense oligodeoxynucleotide(IGF-1R AS ODN), wherein the tumor cells are removed from a resectionsite in the subject using a tissue morselator that does not produce heatat the resection site.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIGS. 1a-1g depict a representative biodiffusion chamber. FIG. 1 a.component parts; FIG. 1 b. assembled chamber; FIG. 1 c. PMMA port plugto seal the chamber; FIG. 1 d. photomicrograph of polyvinylidinefluoride Durapore membrane; FIG. 1 e. overhead and lateral view of theactual chamber; FIG. 1 f. and FIG. 1 g. H & E stained paraffin sectionsof Durapore membranes after explantation; FIG. 1 f. explanted phosphatebuffered saline control chamber from human trial 14379-101; FIG. 1 g.explanted vaccine chamber from human trial 14379-101.

FIGS. 2a-2c depict survival metrics of subjects in Phase I trial (IND14379-101, NCT01550523). FIG. 2a . Overall survival of patients intrial; FIG. 2b . protocol survival with two survival cohorts. Ninepatients died of disease progression while one died of intracerebralhemorrhage and two of sepsis. Overall protocol survival was 48.2 weeksand 9.2 weeks, respectively for longer (N=4) and shorter (N=8) survivalcohorts (log-rank=0.014). FIG. 2c . Excluding one profoundly lymphopenicoutlier and three non-disease-related deaths linear regression revealedhigh correlation between protocol survival and lymphocyte count atenrollment (R²=0.8, p=0.0028).

FIGS. 3a-3d shows radiographic responses with associated physiologicmeasurements. FIG. 3a . Examples of patient imaging from short survivalcohort. Patient TJ11: A-D; Patient TJ10: E-H. A, E: pre-operative T1-gadolinium-enhanced axial images; G: T1-gadolinium-enhanced coronalimage; C: pre-operative axial FLAIR image. B, D, F, H: respective 3month post-operative images. FIG. 3b . Examples of patient imaging fromlonger survival cohort. Patient TJ06: A-D; Patient TJ09: E-H. A, E:pre-operative T1-gadolinium-enhanced axial images; C, F: pre-operativeaxial FLAIR images. B, D, F, H: respective 3 month post-operativeimages. FIG. 3c . Relationship between relative cerebral blood volume intumor v. apparent diffusion coefficient in longer survival cohort; thereis a high correlation between the ADC and rCBV (R²=0.96, p=0.0005). FIG.3d . Relationship between relative cerebral blood volume in tumor v.apparent diffusion coefficient in short survival cohort.

FIGS. 4a-4c depict an examination of the explanted chambers by survivalcohorts. FIG. 4a . Explanted chambers were structurally intact with noviable cells. Outer surfaces of membranes from both C-p and C-v chamberswere coated with CD15+ and CD163+ cells, with dramatically increasednumbers on C-v membranes; FIG. 4b . analysis of factors in chambersbetween survival cohorts revealed significant chamber elevations ofVEGF, PDGF-α, IL-11, CCL5, MCP-3 and MIP-1d in the longer cohort while anumber of soluble cancer markers were significantly elevated in theshort cohort including NSE, osteonectin, and YKL40. Mixture discriminantanalysis independently identified these cohort differences; FIG. 4c .for both cohorts, two chemokines associated with glioma macrophagerecruitment were significantly lower in C-v than other measurablesources. Both Periostin and CCL2 levels were significantly lower thanserum or SN (tumor cell supernatant) values, suggesting elimination ofcells producing these chemokines in the chambers.

FIGS. 5a-5e depict post-vaccination levels of PBMCs and cytokines.Serial measurements of immune effector cell shifts andcytokine/chemokine shifts after vaccination in the post-treatmentperiod; longer survival cohort, (patients TJ03, TJ14, TJ06, TJ09);example of short survival cohort, patient TJ13 (for all other shortsurvival cohorts, see FIG. 6). Rows: FIG. 5a . serial PBMC counts aftervaccination; FIG. 5b . serial assessments of PBMC subpopulationpercentages after vaccination, FIG. 5c . serial levels of CCL21 andCXCL12; FIG. 5d . relationship of absolute CD14+CD16-macrophage countswith MCP-1 (CCL2); note correlation with macrophage levels in FIG. 5b .and CCL2 spike post-operatively. CC12 levels remained significantlyhigher in the short survival cohort (see FIG. 6); FIG. 5e . scaledcomparisons of putative TH-1 cytokine responses after vaccinations(TNF-α×2; CXCL9×350; CXCL10×80). Significant correlations were noted asfollows: TNF-α spikes were highly correlated with CCL2 spikes for bothcohorts (R²=0.99, p=0.003). There was a significant immediateperioperative decrease in CD14+16-cells (p=0.008) not seen in the shortcohort (p=0.78). For the longer cohort only, there was a significantcorrelation between CD4 and CXCL12 (R²=0.62, p<0.0001). Also, a highcorrelation was noted between total monocyte count and CD14+16-monocytelevels (FIGS. 5B and 5D, R²=0.8, p<0.0001) and inverse relationshipsbetween circulating T cell and monocyte numbers (R²=0.66, p<0.0001) werenoted in the longer survival cohort (FIG. 5) without significantdifferences in the short survival cohort (see FIG. 6).

FIGS. 6a-6e depicts post-vaccination levels of PBMC and cytokines inshort cohort patients , (patients TJ01, TJ01, TJ07, TJ08, TJ10, TJ11,TJ12);. Rows: FIG. 6a . serial PBMC counts after vaccination; FIG. 6b .serial assessments of PBMC subpopulation percentages after vaccination(T-cell; B-cell; monocyte); FIG. 6c . serial levels of CCL21 and CXCL12;FIG. 6d . relationship of absolute CD14+CD16-macrophage counts withMCP-1 (CCL2). CC12 levels remained significantly higher in the shortsurvival cohort compared to long survival cohort. FIG. 6e . scaledcomparisons of putative TH-1 cytokine responses after vaccinations(TNF-α×2; CXCL9×350; CXCL10×80). IFN-g is also shown.

FIGS. 7a-7h depict the loss of specific, tumor-promoting monocyte cellpopulations after vaccination. Substantial tumor regression was observedover a 3 month period. FIG. 7a . Monophasic trend for TME IGF-1R+ cells(ordinal scale); in matched pairs cases from initial diagnosis tovaccination (N=5) no significant difference; matched pairs from vaccineto autopsy (N=4) reveals significant decrease in IGF-1R+ cells(p=0.003). FIG. 7b . IGF-1R positive cells in two patients withevaluable paraffin sections from initial diagnosis through vaccine andautopsy (patients TJ06 and TJ10). FIG. 7c . Biphasic trend for TME CD163M2 macrophages with significant increase from diagnosis to recurrence(Aperio five 400× fields per phase of treatment per patient; left plot,matched pairs *p<0.0001, N=6) followed by significant loss fromrecurrence to autopsy after vaccination (right plot, matched pairs*p<0.0001, N=4). FIG. 7d . CD163+ cells in same two patients withevaluable paraffin sections from initial diagnosis through vaccine andautopsy (patients TJ06 and TJ10); increase in CD163 at vaccine v.recurrence (matched pairs, p=0.052) followed by significant decrease inTME CD163 M2 macrophages at autopsy v. vaccine (matched pairs,*p=0.001). FIG. 7e . Significant correlation in the short survivalcohort between peripheral CD163 monocytes and CD163 TAM levelsdocumented at surgery (R²=0.80, p=0.02). FIG. 7f . Non-significantcorrelation between peripheral and TAM CD163 cells in the longer cohort.FIG. 7g . Fluorescence immunohistochemistry photomicrographs fromparaffin sections. A, C: Patient TJ10 at Second surgical resection priorto vaccination and B, D: at autopsy; E-H: autopsy specimens obtainedfrom glioblastoma patients undergoing re-resection after standard ofcare; I, J: untreated, incidentally found post-mortem glioblastoma. FIG.7h . Time course for treatment response in TJ06 from initial diagnosisthrough autopsy. Biphasic occurrence of CD163 cells in the TME withincrease after standard treatment and decrease after vaccination throughautopsy. Loss of CD163 TAMs is associated with increases in both rCBVand ADC values in the tumor. Serum nitrate levels spike after eachvaccination and are associated with concomitant rCBV/ADC increases.

FIGS. 8a-8d depict differentiation of immature monocytes by cytokines orserum from study subject. FIG. 8a . Upregulation of IGF-1R afterpolarization of monocytes with M2 cytokines. M1 macrophages do notupregulate the IGF-1R, ***p=0.0004. FIG. 8b . Differences in monocytesubset distribution after treatment with IGF-1R AS ODN according tomacrophage polarization protocol described in materials and methods.Flow cytometry reveals that IGF-1R AS ODN selectively targets theremoval of M2 macrophages. FIG. 8c . Protocol patient serumdifferentiates immature monocytes into a CD163+phenotype thatco-expresses IGF-1R and PD-L1. IGF-1R AS ODN knocks down thismacrophages population in a dose-dependent fashion over a 100-foldconcentration range. All values are mean fluorescence intensity.Duplicate measurements for each patient serum co-incubation, comparisonof means. ***p<0.0001, **p=0.0001, *p=0.0002, ♦♦♦p=0.0003, ♦♦p=0.0009,♦p=0.009,

p=0.0018,

p=0.026. FIG. 8d . Summary of means in FIG. 8 c.

FIGS. 9a-9d show that compared to standard of care in the first interimanalysis, there were significant improvements in both progression-freesurvival and overall survival. FIG. 9a . Progression-free survival (PFS)of entire study cohort compared to standard of care (SOC); dotted blacklines are 95% confidence interval; FIG. 9b . Overall survival (OS). Inboth cases SOC falls below the lower 95% CI reflecting a significantimprovement; FIG. 9c . PFS by survival cohort at interim analysis; FIG.9d . OS by survival cohort at interim analysis.

FIGS. 10a-10d depict a summary of the Phase 1b study and a comparison ofinterferon-gamma levels to the prior trial and between cohorts withinthe trial. FIG. 10a . Trend of increasing IFN-γ after vaccination in thenewly diagnosed vaccine cohort (p=0.06); FIG. 10b . Significant increasein median IFN-γ in the newly diagnosed vaccine cohort (p=0.02); FIG. 10c. Significant increases in IFN-γ levels in 20 chamber cohorts***p<0.0001, **p<0.006, *p<0.02; FIG. 10d . Rate of diffusion of labeledIGF-1R AS ODN from the biodiffusion chamber over time.

FIGS. 11a-11j depict the effect of fully formulated biodiffusion chamber(both irradiation and exogenously added AS ODN) on pro-inflammatorycytokine production in a naïve mouse model. Luminex analysis ofexplanted mouse chamber contents at 24 hour post implantation filledwith GL261 cells alone; partial formulation with either addition of 2 μgIGF-1R AS ODN, irradiation of GL261 cells with 5 Gy of X-irradiation; orthe fully formulated autologous vaccine (GL261, 2 μg IGF-1R AS ODN, and5 Gy of gamma-irradiation). FIG. 11 a. G-CSF GL261-AS-irr v. GL261-irrp<.0117; FIG. 11b . IL-1a, GL261-AS-irr v. GL261-irr p<0.008; FIG. 11c .IL-1b, GL261-AS-irr v. GL261-irr p<0.0067; FIG. 11 d. IL-2, GL261-AS-irrv. GL261-irr p<0.0002; FIG. 11 e. IL-9, GL261-AS-irr v. GL261-irrp<0.0413; FIG. 11 f. IL-10, GL261-AS-irr v. GL261-irr p<0.0001; FIG. 11g. IL-12(p40), GL261-AS-irr v. GL261-irr p<0.001; FIG. 11h . IL-13,GL261-AS-irr v. GL261-irr p<0.0065; FIG. 11 i. IL-15, GL261-AS-irr v.GL261-irr p<0.0013; FIG. 11j . M-CSF, GL261-AS-irr v. PBS p=0.007.Others tested but not shown: IL-6, GL261-AS-irr v. GL261-irr p<0.0836;GM-CSF, GL261-AS-irr v. GL261-irr p<0.0854; lix, GL261-AS-irr v.GL261-irr p<0.0001; kc, GL261-AS-irr v.GL261-irr p<0.0112; TNF-α,GL261-AS-irr v. GL261-irr p<0.0082; VEGF, GL261-AS-irr v. GL261-irrp<0.0004; lif, GL261-AS-irr v. GL261-irr p<0.0140; IL-7, GL261-AS-irr v.GL261-irr p<0.0038; IL-12(p70) GL261-AS-irr v. GL261-irr p<0.0120;IFN-γ, GL261-AS-irr v. GL261-irr p<0.0290.

FIG. 12 shows titration curves for dendritic cell (DC) activation ofperipheral blood mononuclear cells (PBMC) from two normal subjects (redand blue) by IGF-1R AS ODN; NOBEL antisense 750 μg v. NOBEL sense 75 μg,7.5 DWA antisense 750 μg, 7.5 μg, control, ***p<0.0009; NOBEL antisense75 μg v. NOBEL sense, DWA antisense 7.5 μg.

FIGS. 13a-13b depict in vitro T cell response from contents of fullyformulated chamber utilizing T cells derived from vaccinated mice. FIG.13a . Pro-inflammatory T cell response with DCs primed with antigenretrieved from chambers; **p<0.01 for full formulation in vitro v. noantigen; *p<0.03 for full formulation v. exosomes; FIG. 13b .Pro-inflammatory T cell response with DCs pulsed with antigen retrievedfrom chambers; **p<0.005 for full formulation v. no antigen; *p<0.007for full formulation v. exosomes.

FIGS. 14a-14d are schematic representations of biphasic response toNOBEL antisense dose titration.

FIGS. 15a-15b depict M2 polarization of allogenic monocytes from threenormal subjects with overnight incubation from serum derived from sixdifferent glioma patients. Controls were not incubated with serum.1000-fold dilution curve revealed a decrease of M2 macrophagesco-expressing FIG. 15a . PDL-1 and FIG. 15b . CD163 from 100 μg of NOBELantisense to a significant knockdown at 1 μg. Line in each graph is thegrand mean. 1 μg of NOBEL v. untreated ***p<0.0002.

FIG. 16 depicts comparison of explanted vaccine chamber cytokine levelsv. explanted PBS control chamber. ***p, 0.005; ***p<0.01; ***p<0.02;**p<0.03; *p<0.05.

FIG. 17 is a dose-response curve, showing the biphasic response ofsystemic IGF-1R AS-ODN on inhibition of flank glioma tumor growth. 10⁶GL261 cells were implanted into the flanks of C57BL/6 mice and 20 dayslater, prior to the period when an elevation in circulating CD163positive cells is typically observed, the mice were injectedintraperitoneally with a single 0.75 mg (squares) or 0.075 mg(triangles) dose of NOBEL IGF-1R AS-ODN. The mice were then followed fortumor development. Unvaccinated mice (circles) were used as a control.

FIG. 18 is flow cytometry data showing that systemic IGF-1R AS-ODNtreatment inhibited the accumulation of circulating M2 monocytes. Thedata is expressed as a histogram of cell numbers expressing CD163 (righthand peak) where the red line represents tumor-implanted mice treatedwith PBS (vehicle) and the blue line represents implanted mice treatedwith NOBEL IGF-1R AS-ODN.

FIG. 19 shows tumor incidence in mice implanted in the flank with gliomacells, treated with NOBEL IGF-1R AS-ODN or PBS (vehicle). Tumorincidence between the treated and untreated groups was significantlydifferent (*=p <0.05).

FIG. 20 shows tumor incidence in Tbet deficient mice implanted in theflank with glioma cells, and treated with or without NOBEL IGF-1RAS-ODN. Tumor incidence between the groups was significantly different(*=p <0.05).

FIGS. 21a, 21b, and 21c show pro-inflammatory cytokine levels (pg/ml) inpatient serum after vaccination, pooled from serial blood draws overtime (days 14-42 post-surgery). A significant dose-dependent increase inpro-inflammatory cytokines was observed in patient serum. FIGS. 21d,21e, and 21f depict the relationship (polynomial best fit) between wetweight yield of tumor tissue and cytokine yield by subject. A wet weightyield of tissue of 3 grams produced the highest cytokine yield when,after processing, the cells were distributed among 20 chambers.

FIG. 22 is a schematic of a representative fully formulated biodiffusionchamber. When the chamber is implanted into a patient, antisensemolecules and tumor antigens diffuse through the porous membranes of thechamber, leading to a tumor-specific immune response, decreased M2polarization, and reduction in the number of M2+ cells.

FIG. 23 is a schematic of a representative immunization method. If apatient does not adequately respond to a first round of vaccination, theprocedure is optionally repeated as many times as necessary, sometimesin combination with other treatments.

FIGS. 24a and 24b are Kaplan-Meier curves illustrating medianprogression-free survival (P-FS) and median overall survival (OS) in theintention-to-treat group (N=30), respectively, in human patients havingbrain tumors. (Interim analysis is shown in FIG. 9 above.) The“vaccinated” population is treated with 20 chambers implanted and eachchamber containing 2 μg NOBEL. The “SoC” population is represented usinghistorical data (N=76). The data shows substantially increased survivalboth overall and without progression of the cancer.

FIGS. 25a and 25b are Kaplan-Meier curves illustrating progression-freesurvival and overall survival comparing the same gender and median agein the vaccinated and Standard of Care (SoC) groups respectively. Thedata shows substantially increased survival both overall and withoutprogression of the cancer.

FIGS. 26a and 26b are Kaplan-Meier curves illustrating progression-freesurvival and overall survival when excluding the 5 patients who withdrewfrom treatment and who died from other causes.

FIGS. 27a and 27b are Kaplan-Meier curves illustrating progression-freesurvival and overall survival when excluding the 9 patients who did notcomplete the standard of care (SOC) protocol.

FIGS. 28a, 28b, 28c, 28d . illustrate IFN-γ responses induced based oncell yield in the high vaccine cohort. The data show that the optimumIFN-γ release, based on cell number in the chamber. For FIGS. 28a and28b cell yield is shown in millions of cell. IFN-γ are shown as meanfluorescent intensity (MFI). These data are from the 20-chamber cohortwith each chamber containing 2 μg of NOBEL. Data is presented as apolynomial fit (cubic). FIGS. 28c and 20 db is an extract of the data inFIG. 28a and b showing the substantially linear relationship betweencell number yield versus mean and peak IFNγ response, respectively, upto 20 million cells. The data are presented here as pg/ml.

FIGS. 29a, 29b, and 29c illustrate IFN-γ T cell response relative tochamber formulation regarding IGF-1R antisense pre-incubation prior toencapsulation. FIG. 29a shows the protocol for assessing T-cell responseto tumor antigen. For FIG. 29b Antigen was prepared following thein-vivo clinical chamber paradigm. Approximately 1 million ex-vivo GL261tumor cells were injected into chambers alone or with indicatedantisense concentrations and incubated overnight in the chamber whichwas placed in PBS). The following day, chamber content was extracted andused to pulse naïve dendritic cells. Chamber content which was nottreated overnight with antisense was added to the dendritic cells withthe indicated amounts of NOBEL. Dendritic cells were also left naïve forcontrol. Following an overnight pulse with antigen, dendritic cells werecollected and incubated overnight with T cells from immunized animals ina cell culture plate coated with an ELIPSPOT detection antibody for thecytokine IFNγ. After overnight incubation, the coated plate wasprocessed and developed to enumerate the number of IFNγ producingT-cells which responded to each respective antigen. The data in FIG. 29bshows that tumor antigens were detected in materials recovered fromchambers containing GL261 cells plus antisense but not materials fromchambers cultured with cells alone, even if antisense was added to thematerial when the dendritic cells (DC) were pulsed. The data illustratesthat antisense in chambers with the glioma cells is required to produceimmunostimulatory tumor antigen. For FIG. 29c , GL261 cells were platedin petri dishes and treated overnight with 4 mg NOBEL per 1 millioncells or were left untreated. The cells were then collected and placedinto chambers at 1 million cells and 2 μg NOBEL per chamber. Thechambers were then incubated overnight in PBS and the content wasextracted the following day. Dendritic cells were then pulsed with thechamber content and IFNγ secretion was measured as described above. Thedata illustrates that overnight treatment of GL261 cells with antisenseenhances the amount of antigen produced by these cells as detected by anincrease in the numbers of tumor-immune T cells producing IFNγ.

FIGS. 30a, 30b, 30c, and 30d illustrate the impact levels of expressionof Nestin on efficacy in a mouse model. FIG. 30a shows that high levelof Nestin is associated with improved survival following IGF-1Rantisense treatment. Mice were implanted in the flank with chamberscontaining GL261 cells that expressed high or low levels of the nestinprotein as well as 4 mg antisense. A control group received high nestinexpressing cells alone with no antisense added. The chambers were leftin the flank for 24 hours. The immune response was then allowed todevelop for several weeks and the mice were challenged intra-craniallyon day 35 post-chamber implantation. The immunized mice as well asnon-immune controls were monitored for survival after challenge. FIG.30b shows that a high level of Nestin is associated with better clinicaldisease score. The data shows scored morbidity associated with braintumor progression in orthotopic model after vaccination with fullyformulated chamber by treatment cohort. FIG. 30c and FIG. 30d showincreased production of antibody against GL261 cells associated withhigh levels of nestin expression. FIG. 30c shows day 28 postchamber/pre-intra-cranial. implantation cell ELISA assay data performedwith sera from experimental mice was tested for antibody reactivity toGL261 cell; isolated sera from whole blood taken from the mice. The serawas tested for whole IgG reactivity to GL261 cells with an ELISA assay.FIG. 30d shows cell ELISA data from day 35 post intra-cranialchallenge/71 days post-chamber explanation, using sera from experimentalmice, tested for antibody reactivity to GL261 cells.

DETAILED DESCRIPTION Definitions

All terms not defined herein have their common art-recognized meanings.

As used herein, terms such as “a,” “an,” and “the” include singular andplural referents unless the context clearly demands otherwise.

As used herein, the term “about” when preceding a numerical valueindicates the value plus or minus a range of 10%. For example, “about100” encompasses 90 and 110.

As used herein, the term “autologous” means cells or tissues obtainedfrom the same individual.

As used herein, the term “autologous cancer cell vaccine” refers to atherapeutic produced in part by isolating tumor cells from an individualand processing these tumor cells ex vivo. The cells are thenre-administered to the individual from whom the tumor cells wereisolated. In embodiments, an autologous cancer cell vaccine may compriseadditional components in addition to the tumor cells, such as a bufferand/or antisense nucleic acids. In embodiments, “autologous cancer cellvaccine” may refer to a biodiffusion chamber containing the tumor cellsand one or more additional components. In certain aspects, the“autologous cancer cell vaccine” may be a “fully formulated chamber”also referred to herein as “fully formulated biodiffusion chamber.”

As used herein, the term “fully formulated chamber” or “fully formulatedbiodiffusion chamber” is a biodiffusion chamber that includes autologoustumor cells and other cells included in the tumor microenvironment (TME)that may or may not be treated prior to encapsulation in the chamberwith a first amount of an IGF-1R AS ODN. The cells are encapsulated withexogenous addition of a second amount, for example at least 2 μg, ofIGF-1R AS ODN and the chamber is then irradiated with 5 Gy ofgamma-irradiation.

As used herein, the term “small molecules” includes nucleic acids,peptides, proteins, and other chemicals (such as, for example, cytokinesand growth hormones produced by cells), but does not include cells,exosomes, or microvesicles.

The term “targeting IGF-1R expression” or “targets IGF-1R expression” asused herein refers to administering an antisense nucleic acid that has asequence designed to bind to the IGF-1R.

As used herein, the term “systemic administration” refers to achievingdelivery of a substance throughout the body of a subject. Typicalsystemic routes of administration include parenteral administration,transdermal administration, intraperitoneal administration, intravenousadministration, subcutaneous administration, and intramuscularadministration.

Other administration routes include oral administration, nasaladministration topical administration, intraocular administration,buccal administration, sublingual administration, vaginaladministration, intraheptic, intracardiac, intrapancreatic, byinhalation, and via an implanted pump.

Antisense Molecules

Antisense molecules are nucleic acids that work by binding to a targetedcomplimentary sequence of mRNA by Watson and Crick base-pairing rules.The translation of target mRNA is inhibited by an active and/or passivemechanism when hybridization occurs between the complementary helices.In the passive mechanism, hybridization between the mRNA and exogenousnucleotide sequence leads to duplex formation that prevents theribosomal complex from reading the message. In the active mechanism,hybridization promotes the binding of RnaseH, which destroys the RNA butleaves the antisense intact to hybridize with another complementary mRNAtarget. Either or both mechanisms inhibit translation of a proteincontributing to or sustaining a malignant phenotype. As therapeuticagents, antisense molecules are far more selective and as a result, moreeffective and less toxic than conventional drugs.

The methods and compositions disclosed herein involve the use ofantisense molecules for treating cancer. Typically, the antisensemolecule is an antisense oligodeoxynucleotide (AS-ODN). In someembodiments, the antisense molecule comprises a modified phosphatebackbone. In certain aspects, the phosphate backbone modificationrenders the antisense more resistant to nuclease degradation. In certainembodiments, the modification is a locked antisense. In otherembodiments, the modification is a phosphorothioate linkage. In certainaspects, the antisense contains one or more phosphorothioate linkages.In certain embodiments, the phosphorothioate linkages stabilize theantisense molecule by conferring nuclease resistance, thereby increasingits half-life. In some embodiments, the antisense may be partiallyphosphorothioate-linked. For example, up to about 1%, up to about 3%, upto about 5%, up to about 10%, up to about 20%, up to about 30%, up toabout 40%, up to about 50% up to about 60%, up to about 70%, up to about80%, up to about 90%, up to about 95%, or up to about 99% of theantisense may be phosphorothioate-linked. In some embodiments, theantisense is fully phosphorothioate-linked. In other embodiments,phosphorothioate linkages may alternate with phosphodiester linkages. Incertain embodiments, the antisense has at least one terminalphosphorothioate monophosphate.

In some embodiments, the antisense molecule comprises one or more CpGmotifs. In other embodiments, the antisense molecule does not comprise aCpG motif. In certain aspects, the one or more CpG motifs aremethylated. In other aspects, the one or more CpG motifs areunmethylated. In certain embodiments, the one or more unmethylated CpGmotifs elicit an innate immune response when the antisense molecule isadministered to a subject. In some aspects, the innate immune responseis mediated by binding of the unmethylated CpG-containing antisensemolecule to Toll like Receptors (TLR).

In certain embodiments, the antisense molecule comprises at least oneterminal modification or “cap”. The cap may be a 5′ and/or a 3′-capstructure. The terms “cap” or “end-cap” include chemical modificationsat either terminus of the oligonucleotide (with respect to terminalribonucleotides), and including modifications at the linkage between thelast two nucleotides on the 5′ end and the last two nucleotides on the3′ end. The cap structure may increase resistance of the anti sensemolecule to exonucleases without compromising molecular interactionswith the target sequence or cellular machinery. Such modifications maybe selected on the basis of their increased potency in vitro or in vivo.The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus(3′-cap) or can be present on both ends. In certain embodiments, the 5′-and/or 3′-cap is independently selected from phosphorothioatemonophosphate, abasic residue (moiety), phosphorothioate linkage,4′-thio nucleotide, carbocyclic nucleotide, phosphorodithioate linkage,inverted nucleotide or inverted abasic moiety (2′-3′ or 3′-3′),phosphorodithioate monophosphate, and methylphosphonate moiety. Thephosphorothioate or phosphorodithioate linkage(s), when part of a capstructure, are generally positioned between the two terminal nucleotideson the 5′ end and the two terminal nucleotides on the 3′ end.

In preferred embodiments, the antisense molecule targets the expressionof Insulin like Growth Factor 1 Receptor (IGF-1R). IGF-1R is a tyrosinekinase cell surface receptor that shares 70% homology with the insulinreceptor. When activated by its ligands (IGF-I, IGF-II and insulin), itregulates broad cellular functions including proliferation,transformation and cell survival. The IGF-1R is not an absoluterequirement for normal growth, but it is essential for growth inanchorage-independent conditions that may occur in malignant tissues. Areview of the role of IGF-1R in tumors is provided in Baserga et al.,Vitamins and Hormones, 53:65-98 (1997), which is incorporated herein byreference in its entirety.

In certain embodiments, the antisense molecule is an oligonucleotidedirected against DNA or RNA of a growth factor or growth factorreceptor, such as, for example, IGF-1R.

In certain embodiments, the antisense is a deoxynucleotide directedagainst IGF-1R (IGF-1R AS ODN). The full length coding sequence ofIGF-1R is provided as SEQ ID NO:19 (see, for example, PCT/US2016/26970,which is incorporated herein by reference in its entirety).

In certain embodiments, the antisense molecule comprises nucleotidesequences complementary to the IGF-1R signal sequence, comprising eitherRNA or DNA. The signal sequence of IGF-1R is a 30 amino acid sequence.In other embodiments, the antisense molecule comprises nucleotidesequences complementary to portions of the IGF-1R signal sequence,comprising either RNA or DNA. In some embodiments, the antisensemolecule comprises nucleotide sequences complementary to codons 1-309 ofIGF-1R, comprising either RNA or DNA. In other embodiments, theantisense molecule comprises nucleotide sequences complementary toportions of codons 1-309 of IGF-1R, comprising either RNA or DNA.

In certain embodiments, the IGF-1R AS ODN is at least about 5nucleotides, at least about 10 nucleotides, at least about 15nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 45nucleotides, or at least about 50 nucleotides in length. In someembodiments, the IGF-1R AS ODN is from about 15 nucleotides to about 22nucleotides in length. In certain aspects, the IGF-1R AS ODN is about 18nucleotides in length.

In certain embodiments, the IGF-1R AS ODN forms a secondary structure at18° C., but does not form a secondary structure at about 37° C. In otherembodiments, the IGF-1R AS ODN does not form a secondary structure atabout 18° C. or at about 37° C. In yet other embodiments, the IGF-1R ASODN does not form a secondary structure at any temperature. In otherembodiments, the IGF-1R AS ODN does not form a secondary structure at37° C. In particular embodiments, the secondary structure is a hairpinloop structure.

In some aspects, the IGF-1R AS ODN comprises the nucleotide sequence ofSEQ ID NO:1, or a fragment thereof. In certain embodiments, the IGF-1RAS ODN may have at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 96%, at least about 98%, or 100% identity to SEQ ID NO: 1,or a fragment thereof. In some embodiments, the IGF-1R AS ODN comprisesone or more phosphorothioate linkages.

In certain aspects, the IGF-1R AS ODN consists of SEQ ID NO: 1. NOBEL isan 18-mer oligodeoxynucleotide with a phosphorothioate backbone and asequence complimentary to codons 2 through 7 in the IGF-1R gene. Assuch, NOBEL is an antisense oligonucleotide directed against IGF-1R(IGF-1R AS ODN). The NOBEL sequence, derived as the complimentarysequence of the IGF-1R gene at the 5′ end, is:

-   -   5′-TCCTCCGGAGCCAGACTT-3′.

NOBEL has a stable shelf life and is resistant to nuclease degradationdue to its phosphorothioate backbone. Administration of NOBEL can beprovided in any of the standard methods associated with introduction ofoligodeoxynucleotides known to one of ordinary skill in the art.Advantageously, the AS ODNs disclosed herein, including NOBEL, may beadministered with little/no toxicity. Even levels of about 2 g/kg(scaled) based on mice tests (40 μg in the tail vain) did not revealtoxicity issues. NOBEL can be manufactured according to ordinaryprocedures known to one of ordinary skill in the art.

The anti sense molecule, for example the NOBEL sequence of SEQ ID NO: 1,may also comprise one or more p-ethoxy backbone modifications asdisclosed in U.S. Pat. No. 9,744,187, which is incorporated by referenceherein in its entirety. In some embodiments, the nucleic acid backboneof the antisense molecule comprises at least one p-ethoxy backbonelinkage. For example, up to about 1%, up to about 3%, up to about 5%, upto about 10%, up to about 20%, up to about 30%, up to about 40%, up toabout 50% up to about 60%, up to about 70%, up to about 80%, up to about90%, up to about 95%, or up to about 99% of the antisense molecule maybe p-ethoxy-linked. The remainder of the linkages may be phosphodiesterlinkages or phosphorothioate linkages or a combination thereof. In apreferred embodiment 50% to 80% of the phosphate backbone linkages ineach oligonucleotide are p-ethoxy backbone linkages, wherein 20% to 50%of the phosphate backbone linkages in each oligonucleotide arephosphodiester backbone linkages.

Various IGF-1R antisense sequences are bioactive in some or all of themulti-modality effects of the NOBEL sequence. The 18-mer NOBEL sequencehas both IGF-1R receptor downregulation activity as well as TLR agonistactivity, and further experimentation in mice suggests that bothactivities are necessary for in vivo anti-tumor immune activity. Whilethe AS ODN molecule has anti-tumor activity, the complimentary sensesequence does not, despite also having a CpG motif.

In certain embodiments, the sequence of the antisense is selected fromthe group consisting of SEQ ID NOS 1-14, as shown in Table 1. In someembodiments, the antisense has 90% sequence identity to one or more ofSEQ ID NOS 1-14. In some embodiments, the antisense has 80% sequenceidentity to one or more of SEQ ID NOS 1-14. In some embodiments, theantisense has 70% sequence identity to one or more of SEQ ID NOS 1-14.

TABLE 1 Additional downstream sequences for IGF-1R AS ODN FormulationCorresponds to SEQ ID Sequences with ACGA Motif IGF-1R Codons NO:5′-TCCTCCGGAGCCAGACTT-3′     2-7  1 5′-TTCTCCACTCGTCGGCC-3′    26-32  25′-ACAGGCCGTGTCGTTGTC-3′   242-248  3 5′-GCACTCGCCGTCGTGGAT-3′   297-303 4 5′-CGGATATGGTCGTTCTCC-3′   589-595  5 5′-TCTCAGCCTCGTGGTTGC-3′  806-812  6 5′-TTGCGGCCTCGTTCACTG-3′ 1,033-1,039  75′-AAGCTTCGTTGAGAAACT-3′ 1,042-1,048  8 5′-GGACTTGCTCGTTGGACA-3′1,215-1,221  9 5′-GGCTGTCTCTCGTCGAAG-3′ 1,339-1,345 105′-CAGATTTCTCCACTCGTCGG-3′    27-34 11 5′-CCGGAGCCAGACTTCAT-3′     1-612 5′-CTGCTCCTCCTCTAGGATGA-3′   407-413 13 5′-CCCTCCTCCGGAGCC-3′     4-814

In certain embodiments, the IGF-1R AS ODN comprises the nucleotidesequence of any one of SEQ ID NOs:1-14, or fragments thereof. In certainembodiments, the IGF-1R AS ODN may have at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 98%, or 100%identity to any one of SEQ ID NOs: 1-14, or fragments thereof.

In some embodiments, the antisense molecule downregulates the expressionof genes downstream of IGF-1R pathway in a cell. In certain aspects, thedownstream gene is hexokinase (Hex II). In some embodiments, theantisense molecule downregulates the expression of housekeeping genes inthe cell. In some aspects, the housekeeping gene is L13.

In certain aspects, the IGF-1R AS ODN is chemically synthesized. Incertain embodiments, the IGF-1R AS ODN is manufactured by solid phaseorganic synthesis. In some aspects, the synthesis of the IGF-1R AS ODNis carried out in a synthesizer equipped with a closed chemical columnreactor using flow-through technology. In some embodiments, eachsynthesis cycle sequence on the solid support consists of multiplesteps, which are carried out sequentially until the full-length IGF-1RAS ODN is obtained. In certain embodiments, the IGF-1R AS ODN is storedin a liquid form. In other embodiments, the IGF-1R AS ODN is lyophilizedprior to storing. In some embodiments, the lyophilized IGF-1R AS ODN isdissolved in water prior to use. In other embodiments, the lyophilizedIGF-1R AS ODN is dissolved in an organic solvent prior to use. In yetother embodiment, the lyophilized IGF-1R AS ODN is formulated into apharmaceutical composition. In some aspects the pharmaceuticalcomposition is a liquid pharmaceutical composition. In other aspects,the pharmaceutical composition is a solid pharmaceutical composition.Additional antisense nucleic acids are also described in U.S.Publication No. 2017/0056430, which is incorporated herein by referencein its entirety.

Autologous Cancer Cell Vaccine

Introduction

Immunotherapy is currently used to target hematologic malignancies withone common cellular antigen. Unfortunately, solid tumors are far morecomplex, representing epigenetic progression of genetic changes to amalignant state with an unidentifiable number of tumor-specific targets.Even more challenging, within a WHO diagnostic cancer group there existsmarked variations in tumor phenotypes. An autologous cell vaccine wouldencompass all such variations and all such targets and represent anideal subject-specific immunotherapy for solid tumor cancers. Anautologous cancer cell vaccine however, cannot be derived from primarycell cultures because serial passages alter the tumor phenotype thusdiminishing the array of tumor-specific antigens. This would alsorequire impossible lot-release qualification at each passage. Thepresent disclosure eliminates these concerns by plating freshlyresected, morselized tumor cells and reimplanting them within 24 hoursas a depot antigen, as shown in FIG. 22. In certain aspects, theexcellent results achieved herein are obtained by ensuring that anappropriate number of cells are present in the chamber(s), among otherspecifics described herein.

Previous studies have designed autologous cell vaccine through the useof antigen presenting cells, instead of autologous tumor cells. In thisparadigm, a subject's monocytes are collected from a pre-treatmentplasma leukopheresis and differentiated into autologous dendritic cells(DC) ex vivo. The dendritic cells are then presented with the subject'stumor crude lysate inducing DC activation/maturation, and at a latertime point, the matured dendritic cells, now cross-primed with tumorantigens are injected in the subject as a DC vaccine. Ex vivodifferentiation, however, is missing a number of key stimulatorycomponents only occurring in vivo. In addition, differentiation of DCsfrom hematopoietic precursors requires extensive in vitro manipulationswith labor-intensive cell processing in expensive facilities. Thepresent disclosure obviates these concerns by providing an endogenous DCmaturation process and an immunomodulatory and immunostimulatoryantisense oligodeoxynucleotide (AS-ODN) that promotes the development ofan appropriate immune response. More specifically, the presentdisclosure provides a biodiffusion chamber comprising dispersed tumorcells derived from the patient and irradiated antisense molecules, whichis implanted into the patient for therapeutically effective time.Without being bound by any theory, it is thought that the combination ofirradiated tumor cells, antisense, and biodiffusion chamber act inconcert to simulate the local immune response, and enhance the responseby reducing or eliminating M2 cells, preventing dampening of the immunesystem.

Thus, the present disclosure shows that an irradiated, implantablebiodiffusion chamber comprising freshly resected tumor cells and IGF-1RAS ODN safely serves as an effective, subject-specific autologous cellvaccine for cancer immunotherapy. As such, the use of the claimedimplantable biodiffusion chamber to mount an immune response thatselectively targets tumor cells in a subject provides a new andsignificant approach for the treatment of cancer, especially GBM.

Biodiffusion Chamber

A representative diffusion chamber comprises a chamber barrel having twoends, a first end and a second end. In embodiments, the biodiffusionchamber is a small ring capped on either side by a porous,cell-impermeable membrane, such as the Duropore membrane manufactured byMillipore Corporation. Optionally, one of the ends may be closed off aspart of the chamber body leaving only one end open to be sealed usingthe porous membrane. The membranes can be made of plastic, teflon,polyester, or any inert material which is strong, flexible and able towithstand chemical treatments. The chamber can be made of any substance,such as and not limited to plastic, teflon, lucite, titanium, Plexiglassor any inert material which is non-toxic to and well tolerated byhumans. In addition, the chambers should be able to survivesterilization. In some aspects, the diffusion chambers are sterilizedwith ethylene oxide prior to use. Other suitable chambers are describedin U.S. Prov. No. 62/621,295, filed Jan. 24, 2018, U.S. Pat. No.6,541,036, PCT/US16/26970, and U.S. Pat. No. 5,714,170, which are eachincorporated herein by reference in their entirety.

In certain embodiments, the membrane allows passage of small moleculesbut does not allow passage of cells (i.e., the cells cannot leave orenter the chamber). In some aspects, the diameter of the pores of themembrane allows nucleic acids and other chemicals (such as, for example,cytokines produced by cells) to diffuse out of the chamber, does notallow passage of cells between the chamber and the subject in which itis implanted. The biodiffusion chambers useful in the present disclosureinclude any chamber which does not allow passage of cells between thechamber and the subject in which it is implanted, provided however, thatthe chamber permits interchange and passage of factors between thechamber and the subject. Thus, in certain aspects, the pore size has acut-off that prevent passage of materials that are greater than 100 μm³in volume into and out of the chamber. In some embodiments, the pores ofthe membrane have a diameter of about 0.25 μm or smaller. For example,the pores may have a diameter of about 0.1 μm (see FIG. 1). Inparticular aspects, the pores range in diameter from 0.1 μm to 0.25 μm.See also, Lange, et al., J. Immunol., 1994, 153, 205-211 and Lanza, etal., Transplantation, 1994, 57, 1371-1375, each of which is incorporatedherein by reference in their entireties. This pore diameter prevents thepassage of cells in or out of the chamber. In certain embodiments,diffusion chambers are constructed from 14 mm Lucite rings with 0.1 μmpore-sized hydrophilic Durapore membranes (Millipore, Bedford, Mass.).

In certain embodiments, a biodiffusion chamber comprises a membrane thatallows the IGF-1R AS ODN to diffuse out of the chamber. In someembodiments, about 50% of the IGF-1R AS ODN diffuses out of the chamberin about 12 hours, about 60% of the IGF-1R AS ODN diffuses out of thechamber in about 24 hours, about 80% of the IGF-1R AS ODN diffuses outof the chamber in about 48 hours, and/or about 100% of the IGF-1R AS ODNdiffuses out of the chamber in about 50 hours.

In an exemplary approach, to assemble the biodiffusion chamber, a firstporous membrane is attached to one side of a first diffusion chamber,using glue and pressure to create a tight seal. A second porous membraneis similarly attached to a second diffusion chamber ring. The membranescan be secured in position with rubber gaskets which may also provide atighter seal. The diffusion chamber rings are left overnight (minimum 8hours) to dry. Then, the first diffusion chamber ring and the seconddiffusion chamber ring are attached to one another using glue and leftovernight (minimum 8 hours) to dry. In a preferred embodiment, the firstchamber ring and second chamber ring joining process comprises using 2dichloroethane as a solvent to facilitate adhesion between the tworings. See, for example, FIG. 22 showing two porous membranes. In analternative approach, the chamber may have only one side that contains aporous membrane.

On the barrel portion of the chamber, one or more openings (e.g. ports)are provided which can be covered by a cap which is accessed fromoutside of the subject's body once the chamber is implanted, thusallowing the diffusion chamber to be refilled. The openings allow formultiple and sequential sampling of the contents, without contaminationand without harming the subject, therefore significantly reducing thenumber of implantation procedures performed on the subject. Beforeimplantation into the patient, the one or more openings may be sealedwith bone wax, a port plug or cap made from, for example, PMMA. The capcan be a screw-on type of self-sealing rubber and fitted to the opening.In some configurations, the diffusion chamber may contain two or moreinjection openings or ports. Sampling of the chamber contents can beperformed by accessing the opening by removing the cap on the outside ofthe subject's body and inserting an ordinary needle and syringe. In someembodiments, the chamber may further include a removal device. Such adevice facilitates removal of the chamber from the patient.

In embodiments, the chamber serves as an antigen depot designed so thattumor antigens diffuse out of the chamber for the purpose of promoting atherapeutic host immune response. Exogenous IGF-1R AS ODN and ex vivoirradiation promote a pro-inflammatory response. This formulation isassociated with clinical and radiographic improvements, prolongedsurvival on protocol, and represents a novel autologous cell vaccinethat includes an exogenous active pharmaceutical ingredient (API) andradiation that we interpret as inducing or enhancing tumor immunityeffect. Furthermore the addition of low concentration of the IGF-1R ASODN is critical to a pro-inflammatory response (FIG. 12).

In certain embodiments the disclosure provides a biodiffusion chamberfor implantation into a subject suffering from cancer comprising: (a)tumor cells; and (b) an effective amount of an antisense molecule. Inother embodiments is provided a method for treating cancer in a subjectcomprising: (a) obtaining a biodiffusion chamber comprising tumor cellsand an effective amount of an antisense nucleic acid; (b) irradiatingthe biodiffusion chamber and contents; and (c) implanting the irradiatedbiodiffusion chamber into the subject for a therapeutically effectivetime.

In certain embodiments, the IGF-1R AS ODN is present in the biodiffusionchamber in an amount ranging from about 0.5 μg to about 10 μg. Incertain aspects, the IGF-1R AS ODN is present in an amount ranging fromabout 1 μg to about 5 μg per chamber, or from about 2 μg to 4 perchamber. In specific aspects, the IGF-1R AS ODN is present in an amountof about 2 μg per chamber. In specific aspects, the IGF-1R AS ODN ispresent in an amount of about 4 μg per chamber. Without being bound bytheory it is thought that these levels promote an enhanced Th1 responsein a subject, while avoiding an M2 immunostimulatory response in thesubject.

In certain embodiments, the tumor cells are not treated with an IGF-1RAS ODN prior to encapsulation in the chamber. Typically, however, thetumor cells are treated with an IGF-1R AS ODN prior to encapsulation inthe chamber. The time for treating the cells pre-encapsulation may vary.For example, the tumor cells may be treated ex vivo with an IGF-1R ASODN immediately before encapsulation, for up to about 4 hours, for up toabout 6 hours, for up to about 8 hours, for up to about 12 hours or forup to about 18 hours. Typically, the tumor tissue may be treated ex vivofor about 12 hours to about 18 hours pre-encapsulation. Conveniently,the cells may be encapsulated after a pre-treatment lasting up toovernight. Without being bound by theory, it is thought that thepre-encapsulation treatment plays a desirable role in stimulatingproduction of tumor antigen.

The amount of IGF-1R AS ODN used for the pre-encapsulation treatment maybe in a range of about 1 mg to 8 mg per million cells; for example,about 2 mg to about 6 mg per million cells, about 3 mg to about 5 mg permillion cells. Typically the amount of IGF-1R AS ODN used for treatmentprior to encapsulation is about 4 mg per million cells.

In some embodiments, the IGF-1R AS ODN for ex vivo treatment of thetumor cells is used at a concentration ranging from about at least 2mg/ml to at least about 5 mg/ml. In certain aspects, the IGF-1R AS ODNis used at a concentration of at least 4 mg/ml. In specific embodiments,the IGF-1R AS ODN is used at a concentration of 4 mg/ml.

In certain embodiments, the IGF-1R AS ODN used to treat tumor cells exvivo and the IGF-1R AS ODN present in the chamber are the same. In otherembodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo and theIGF-1R AS ODN present in the chamber are different. In certainembodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo is atleast about 5 nucleotides, at least about 10 nucleotides, at least about15 nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 45nucleotides, or at least about 50 nucleotides in length. In someembodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo is fromabout 15 nucleotides to about 22 nucleotides in length. In certainaspects, the IGF-1R AS ODN used to treat tumor cells is about 18nucleotides in length.

In certain embodiments, the IGF-1R AS ODN used to treat tumor cells exvivo forms a secondary structure at 18° C., but does not form asecondary structure at about 37° C. In other embodiments, the IGF-1R ASODN used to treat tumor cells does not form a secondary structure atabout 18° C. or at about 37° C. In yet other embodiments, the IGF-1R ASODN used to treat tumor cells ex vivo does not form a secondarystructure at any temperature. In other embodiments, the IGF-1R AS ODNused to treat tumor cells does not form a secondary structure at 37° C.In particular embodiments, the secondary structure is a hairpin loopstructure.

In some aspects, the IGF-1R AS ODN used to treat tumor cells comprisesthe nucleotide sequence of SEQ ID NO:1, or a fragment thereof. Incertain embodiments, the IGF-1R AS ODN used to treat tumor cells mayhave at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 98%, or 100% identity to SEQ ID NO: 1, or a fragmentthereof. In certain aspects, the IGF-1R AS ODN used to treat tumor cellsis SEQ ID NO: 1.

After the tumor cells are treated with the AS-ODN for a period of time,the AS-ODN is removed and fresh AS-ODN is added to the chamber, which isthen irradiated prior to implantation into a subject. In certainaspects, the biodiffusion chamber is treated with gamma irradiation atan amount of about 1 Gy, about 2 Gy, about 4 Gy, about 5 Gy, about 6 Gy,about 10 Gy, or up to about 15 Gy. In certain aspects, the dose ofradiation is not more than about 5 Gy. In other aspects, the dose ofradiation is at least about 5 Gy. In some aspects, the dose of radiationis 5 Gy. In certain embodiments, the biodiffusion chamber may beirradiated at least once, at least twice, at least three times, at leastfour times, or at least five times. In some embodiments, the chamber isirradiated less than about 24 hours prior to implantation into asubject. In other embodiments, chamber is irradiated about 24 hoursprior to implantation into the subject. In yet other embodiments, thechamber is irradiated at least about 24 hours prior to implantation intothe subject. In still other embodiments, the chamber is irradiated notmore than about 48 hours prior to implantation into the subject. In yetother embodiments, the chamber is irradiated at least about 48 hoursprior to implantation into the subject.

While the tumor cells are typically killed prior to implantation; forexample by radiation, the cells need not be killed and indeed it may beadvantageous to maintain the cells in an alive state to promote releaseof antigen. Thus, in certain embodiments, the cells may not beirradiated prior to implantation. For safety purposes, however, it isdesirable to prevent release of live tumor cells into the subject.

Tumor cells can be placed in a diffusion chamber in varying numbers. Incertain embodiments, about 1×10⁴ to about 5×10⁶ tumor cells are placedin each diffusion chamber. In other embodiments, about 1×10⁵ to about1.5×10⁶ tumor cells are placed in the diffusion chamber. In yet otherembodiments, about 5×10⁵ to 1×10⁶ tumor cells are placed in the chamber.with a subject can be used. We have discovered that the number of tumorcells can impact the subjects' anti-tumor response and that anappropriate range should be selected to increase the chance to obtainthe desired results. FIG. 28 shows data from patients implanted with 20chambers and shows cell yield (millions of cells) corresponding toimmune response. The anti-tumor immune response is optimal in a range ofabout 750,000 to about 1,250,000 cells in a chamber, with a peak atabout 1 million cells/chamber. Multiple chamber containing irradiatedtumor cells are administered and to maintain the optimal immune theresponse the number of cells/chamber is preferably maintained within therange. Preferably, the tumor cells are intact and not autolyzed orotherwise damaged as described herein.

In certain embodiments, it may be preferable to maintain the ratio ofcells to AS ODN in a chamber. Thus, in certain aspects a chambers maycontain about 2 μg of AS ODN and between 750,000 and 1,250,000 cells;for example 1,000,000 cells. The ratio of cells to AS ODN may thus be ina range from about 3.75×10⁵ to about 6.25×10⁵ per μg AS ODN; forexample, about 5.0×10⁵ cells per μg. Thus, in a typical patientreceiving 20 chambers the total dose of AS ODN is about 40 μg.

Typically, administration will be in a chamber as described herein;however, in certain aspects, the irradiated cells and IGF-1R AS ODN maybe co-administered to the subject without being contained physicallytogether in the chamber or another container. In certain methods usingthis approach, the irradiated cells IGF-1R AS ODN thus disperse,diffuse, or are metabolized in the body limited by the physiology of thesubject. Thus, in certain aspects, e.g. the tumors cells for use may beprepared as described herein for the chamber and administered with theIGF-1R AS ODN but the administration may be not contained within aphysical container. Such administration is typically intramuscular.

Tumor Tissue Preparation for Chamber

Tumor cells for use in the autologous vaccination are surgically removedfrom the subject. In embodiments, the tumor cells are removed from thepatient using a tissue morselator. The extraction device preferablycombines a high-speed reciprocating inner cannula within a stationaryouter cannula and electronically controlled variable suction. The outercannula has a diameter of 1.1 mm, 1.9 mm, 2.5 mm, or 3.0 mm, and alength of 10 cm, 13 cm, or 25 cm. The instrument also relies on aside-mouth cutting and aspiration aperture located 0.6 mm from the bluntdesiccator end. The combination of gentle forward pressure of theaperture into the tissue to be removed and suction draws the desiredtissue into the side aperture, allowing for controlled and precisetissue resection through the reciprocal cutting action of the innercannula. A key feature is the absence of a rotation blade; this avoidsdrawing unintended tissue into the aperture. An example of a suitabledevice is the Myriad® tissue aspirator (NICO Corporation® Indianapolis,Ind.), a minimally invasive surgical system which may be used for theremoval of soft tissues with direct, microscopic, or endoscopicvisualization. The shaved tissue is suctioned, gathered in to acollection chamber, and is collected in a sterile tissue trap. Duringcollection of the tissue in the sterile tissue trap, blood is removedfrom the preparation. Preferably, the sterile trap contains a collectiondish at the bottom of the trap and a stem that provides access to thetrap. The trap structure may also contain an inner ladle-shapedstructure that is removable from the trap to facilitate tissue removalfrom the trap.

Preferably, the morselator generates no heat at the resection site oralong its shaft, and requires no ultrasonic energy for tissue removal.Thus, in particular embodiments, the tumor tissue is morselized tumortissue (i.e. tumor shaved tissue obtained by side-mouth cutting in theabsence of heat, and optionally in the absence of ultrasonic treatment).Advantageously, the aspirator-extract and morselized tissue has higherviability than tissue removed by other methods. It is believed that theextraction process maintains higher tumor cell viability in part due torestricting exposure of the tumor cells to high temperatures duringremoval. For example, the methods herein do not expose tumor cells toabove 25° C. during removal. Thus, the cells are not exposed totemperatures above body temperature, i.e., about 37 ° C.

The amount of tumor tissue obtained from the subject may vary.Preferably, the amount is at least 1, at least 2, at least 3 grams or atleast 4 grams of wet tumor tissue is obtained from the patient. Thetissue is removed from the sterile tissue trap and disaggregated bypipetting with a sterile pipette to break up large tissue fragments. Thedisaggregated cell suspension is then placed onto sterile tissue cultureplates in serum-containing media, and incubated in a tissue cultureincubator. This plating step serves to enrich the desired functionalcells by adherence, and also helps to remove debris from thepreparation. Thus, the tumor cells used in treatments described hereinpreferably consist essentially of, or consist of, adherent cells fromthe tumor tissue.

After a predetermined incubation time (e.g., 6, 12, 24, or 48 hours),the cells are removed from the plates. The cells may be removed byscraping, by chemical methods (e.g. EDTA) or by enzymatic treatment(e.g. trypsin). The cells are placed into one or more diffusionchambers. In some embodiments, the cells are split between 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more diffusion chambers. Often, 20 chambersare used. In some embodiments, each diffusion chamber contains an equalnumber of cells. In some embodiments, a first diffusion chamber containsmore cells than a second chamber.

In some embodiments, the cells are sorted before being placed in thechamber. In some embodiments, the cells are enriched by selecting forone or more cellular markers before being placed in the chamber. Theselection may be performed, for example, using beads or by cell sortingtechniques known to those of skill in the art. In some embodiments, thecells placed into the chamber are enriched for one or more markers.

In some embodiments, implantation of the biodiffusion chamber for atherapeutically effective time reduces or eliminates return of thecancer in the subject. In certain aspects, implantation of thebiodiffusion chamber causes a reduction of tumor volume associated withthe cancer in the subject. In yet other embodiments, implantation of thebiodiffusion chamber for a therapeutically effective time induceselimination of the tumor in the subject. In some embodiments,implantation of the chamber inhibits regrowth of the tumor for at least3 months, at least 6 months, at least 12 months, at least 36 month, orindefinitely.

The biodiffusion chamber can be implanted in a subject in the followingnon-limiting ways: subcutaneously, intraperitoneally, andintracranially. In certain embodiments, the diffusion chamber(s) isimplanted into an acceptor site of the body having good lymphaticdrainage and/or vascular supply such as the rectus sheath. In otherembodiments, a refillable chamber can be employed such that thediffusion chamber can be re-used for treatments and emptied followingtreatments. In certain aspects, a plurality of diffusion chambers,preferably between 5 and 20, can be used in a single subject.

In certain embodiments, at least about 1, at least about 2, at leastabout 3, at least about 4, at least about 5, at least about 10, at leastabout 15, at least about 20, at least about 25, at least about 30, atleast about 35, at least about 40, at least about 45, or at least about50 chambers are implanted into the subject. In some embodiments, 10-20chambers are implanted into the subject. Preferably, about 20 chambersare implanted into the subject. In certain embodiments, the tumor cellsare divided equally among each chamber.

Typically, the chamber is removed after period of time. For example, thechamber may be implanted in the subject for about 24 hours, about 48hours, about 72 hours, or about 96 hours. Implantation for about 48hours is associated with beneficial therapeutic outcomes. Accordingly,the preferred time of implantation is about 48 hours. In certainembodiments, the vaccination procedure is performed one time perpatient. In other embodiments, the vaccination procedure is performedmultiple times per patient. In embodiments, the vaccination procedure isperformed two times, three times, four times, five times, six times,seven times, or eight times in a single patient. In embodiments, thevaccination is repeated every 7, 14, or 28 days, or every 1, 3, or 6months for a given period of time. In further embodiments, thevaccination procedure is repeated periodically until the patient is freeof cancer.

Without being bound by theory, it is thought that implantation of thebiodiffusion chamber causes elimination or reduction of M2 cells at ornear the implantation site such that an immune response against tumorantigens diffusing out from the chamber is achieved. In certain aspects,elimination or reduction of M2 cells at the implantation site leads toenhanced presentation of autologous tumor antigens by antigen-presentingcells (APC) to CD4 T cells leading to production of interferon-gamma(IFNγ) and the induction of type 1 tumor immunity. In certain aspects,the production of IFNγ by tumor antigen-specific CD4 T cells and theanti-M2 effects of IGF-1R AS ODN drive type 1 anti-tumor immunity andthe loss of anti-inflammatory M2 cells from the circulation and tumormicroenvironment indirectly interfering with tumor growth. In someaspects, the production of IFNγ by tumor antigen-specific CD4 T cellsand the anti-M2 effects of IGF-1R AS ODN unleashes effector-mediateddamage to the tumor cells and tumor microenvironment (M2 cells) andinitiates the longer process of programming memory T cells recognizingtumor antigens. In certain embodiments, the anti-tumor adaptive immuneresponse sustains continued tumor regression.

Optionally, the cells introduced into the chamber may be enriched forcertain cell types. Nestin a, cytoskeleton-associated class VIintermediate filament (IF) protein, has traditionally been noted for itsimportance as a neural stem cell marker. We have discovered that incertain brain tumor samples, cells positive for nestin (nestin+cells)are enriched compared to benign tissue, and that this associatedcorresponds to improved therapeutic response. Thus, in certain aspects,a subject's tumor can be biopsied to assess the degree of nestinexpression, and therefore, in certain aspects, the chamber cells areenriched Nestin-positive (“+”) cells compared to benign tissue. Withoutbeing bound by theory, it is thought that nestin provides a markerassociated with antigens suitable useful in producing an anti-tumorimmune response. Accordingly, the cells implanted into the chamber maybe enriched for nestin+cells compared to the tumor cell population as awhole when extracted from the subject. FIG. 30 illustrates the enhanceimmune response obtained when the tumor sample used to stimulate aresponse is enriched with Nestin.

Systemic Administration

As an alternative to, or supplement to, implantation of the chambers,IGF-1R AS ODN may be administered systemically. Thus, in embodiments,the IGF-1R AS ODN is provided in a pharmaceutical composition forsystemic administration. In addition to the IGF-1R AS ODN, thepharmaceutical composition may comprise, for example, saline (0.9%sodium chloride). The composition may comprise phospholipids. In someaspects, the phospholipids are uncharged or have a neutral charge atphysiologic pH. In some aspects, the phospholipids are neutralphospholipids. In certain aspects, the neutral phospholipids arephosphatidylcholines. In certain aspects, the neutral phospholipids aredioleoylphosphatidyl choline (DOPC). In some aspects, the phospholipidsare essentially free of cholesterol.

In some aspects, the phospholipids and oligonucleotides are present at amolar ratio of from about 5:1 to about 100:1, or any ratio derivabletherein. In various aspects, the phospholipids and oligonucleotides arepresent at a molar ratio of about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1,35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1,95:1, or 100:1. In some aspects, the oligonucleotides and phospholipidsform an oligonucleotide-lipid complex, such as, for example, a liposomecomplex. In some aspects, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of the liposomes are less than 5 microns indiameter. In various aspects, the composition further comprises at leastone surfactant, such as, for example, polysorbate 20. In some aspects,at least about 5% of the total liposomal antisense drug product consistsof surfactant and at least about 90% of the liposomes are less than 5microns in diameter. In some aspects, at least about 15% of the totalliposomal antisense drug product consists of surfactant and at leastabout 90% of the liposomes are less than 3 microns in diameter. In someaspects, the population of oligonucleotides are incorporated in thepopulation of liposomes.

In some aspects the pharmaceutical composition is a liquidpharmaceutical composition. In other aspects, the pharmaceuticalcomposition is a solid pharmaceutical composition.

Dosages for systemic administration of the antisense in human subjectsmay be about 0.025 g/kg, about 0.05 g/kg, about 0.1 g/kg, about 0.15g/kg, or about 0.2 g/kg. In certain embodiments, the dosage for systemicadministration may be from 0.025 g/kg to 0.2 g/kg. In some embodiments,the dosage is about 0.2 g/kg. In other embodiments, the dosage is from0.004 g/kg to 0.01 g/kg. In other embodiments, the dosage is less than0.01 g/kg. In further embodiments, the dosage is not between 0.01 g/kgto 0.2 g/kg. In certain aspects, the antisense is supplied as alyophilized powder and re-suspended prior to administration. Whenresuspended the concentration of the antisense may be about 50 mg/ml,about 100 mg/ml, about 200 mg/ml, about 500 mg/ml, about 1000 mg/ml, ora range between those amounts.

In certain embodiments, the AS ODN may be administered systemicallypre-operatively; for example prior to surgery to reduce tumor burden.For example, the AS ODN may be administered up to 24 hours, up to 36hours, up to 48 hours or up to 72 hours before surgery. In particularaspects, the pharmaceutical composition may be administered about 48 toabout 72 hours before surgery. Typically, in such circumstances, theadministration is by intravenous bolus.

Combination Therapies

Historically, cancer therapy has involved treating subjects withradiation, with chemotherapy, or both. Such approaches havewell-documented challenges. Advantageously, however, the chamberimplantation methods disclosed herein may be used to treat a subjecthaving cancer as a monotherapy. Thus it is preferable that the methodsdisclosed herein do not include chemotherapy or radiation therapy.Notwithstanding the excellent effect achieved by monotherapy approachesherein, however, it may be beneficial under certain circumstances tocombine the chamber methods with other therapies; for example, radiationtherapy. In certain embodiments, the radiation therapy includes, but isnot limited to, internal source radiation therapy, external beamradiation therapy, and systemic radioisotope radiation therapy. Incertain aspects, the radiation therapy is external beam radiationtherapy. In some embodiments, the external beam radiation therapyincludes, but is not limited to, gamma radiation therapy, X-ray therapy,intensity modulated radiation therapy (INERT), and image-guidedradiation therapy (WIRT). In certain embodiments, the external beamradiation therapy is gamma radiation therapy. Radiation may beadministered before chamber implantation or after implantation; forexample, as a salvage therapy. Typically, such salvage therapyapproaches are not implemented until the cancer is determined to havereturned.

Thus, in certain combination approaches, both the chamber methods, andthe systemic methods and compositions, described herein may be used inthe same subject, alone or in combination with radiation orchemotherapy. In the combination approaches described herein, thechamber implantation is preferably used as a first-line therapy. Usingthe chamber implantation first is desirable because the subject's immunesystem can be inhibited by other therapies, reducing the therapeuticbenefit of the chamber implantation.

Optionally, systemic administration may be performed prior to chamberimplantation. Such an approach can be used to enhance the subjectsimmune system, as a priming approach. The priming approach may beespecially advantageous where prior therapy has resulted in the subjecthaving a compromised immune system.

When systemic administration is used in combination, the AS ODN may besystemically administered at least 2 weeks, at least 1 week, at least 3days, or at least 1 day prior to treatment of the patient using anautologous cancer cell vaccine. In other embodiments, the AS ODN may besystemically administered at least 1 day, at least 3 days, at least 1week, or at least 2 weeks following treatment of the patient using anautologous cancer cell vaccine; i.e. the chamber.

Optionally, the subject may be revaccinated with chambers using themethods described here subsequent to the first vaccination. A second orfurther additional vaccination may use tumor cells taken from thesubject during the tissue removal and stored. Optionally, the second orfurther additional vaccination may use fresh tumor tissue removed fromthe subject and treated as described herein. Any tumor remaining in thesubject may express the same antigens and thus act as a depot, providingfor re-stimulation. However, recurring tumors may develop new antigensand thus provide additional options to stimulate an anti-tumor response.A subsequent vaccination may be after the first treatment is completeand the tumor has recurred or if the subject has not responded to thefirst treatment.

Subjects for Treatment with the IGF-1R AS ODN

Suitable subjects are animal with cancer; typically, the subject is ahuman. While brain cancers, such as glioblastoma, benefit particularlyfrom this methods disclosed herein, the methods apply to cancergenerally. Accordingly, the disclosure provides methods of treatingcancers, including those selected from the group consisting of: glioma,astrocytoma, hepatocarcinoma, breast cancer, head and neck squamous cellcancer, lung cancer, renal cell carcinoma, hepatocellular carcinoma,gall bladder cancer, classical Hodgkin's lymphoma, esophageal cancer,uterine cancer, rectal cancer, thyroid cancer, melanoma, colorectalcancer, prostate cancer, ovarian cancer, and pancreatic cancer. Inspecific embodiments, the cancer is a glioma. In certain aspects, theglioma is recurrent malignant glioma. In some embodiments, the cancer isan astrocytoma. In certain embodiments, the subject who is a candidatefor treatment is suffering from WHO grade II, WHO grade III, or WHOgrade IV tumor. In some aspects, the tumor is an astrocytoma. In certainembodiments, the tumor is selected from grade II astrocytoma, AIII (IDHIR132H mutant grade III astrocytoma), AIII-G (IDH1 wild-type grade IIIwith characteristics of glioblastoma multiforme astrocytoma), or gradeIV astrocytoma.

Grade IV astrocytoma is the highest grade glioma and is synonymous withglioblastoma (GBM). With a yearly incidence of 3 or 4 per 100,000 GBM isthe most common malignant primary brain tumor in adults. Standard ofcare therapy—typically a combination of radiotherapy and chemotherapyusing Temozolomide—does not work well and the outcome of GBM patientsremains poor with a median life expectancy of 15-17 months.Advantageously, the methods here may be used to treat newly diagnosedbrain cancers and may also be used to treat recurrent glioblastoma; forexample, in patients previously treated with standard of care therapy.Thus, in certain aspects, the subject may be a newly diagnosed GBMsubject or a recurrent GBM subject. The subject is preferably one whohas not been previously treated with any therapeutic approaches that areimmunosuppressive. In particular aspects, eligible subjects are over 18years of age and have a Karnofsky score of 60 or above. Optionally, thesubjects do not have bihemispheric disease and/or do not have anautoimmune disease.

Optionally, a subject who is a candidate for treatment may be identifiedby performing a tumor biopsy on the subject. In some embodiments, tumorsfrom the subject are assayed for the presence of monocytes. In certainaspects, the monocytes include, but are not limited to, CD11b+, CD14+,CD15+, CD23+, CD64+, CD68+, CD163+, CD204+, or CD206+ monocytes. Thepresence of monocytes in the tumors may be assayed usingimmunohistochemistry. In certain embodiments, a subject who is acandidate for treatment shows CD163+ M2 cells greater than about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, or about 50% of the subjects total peripheral blood mononuclearcells (PBMCs). In certain aspects, the subject shows CD163+ M2 cellsgreater than about 20% of the subject's total PBMCs.

In yet other embodiments, a subject who is a candidate for treatment isidentified by the presence of one or more cytokines in the serum of thesubject. These cytokines include, without limitation, CXCL5, CXCL6, andCXCL7, IL6, IL7, IL8, IL10, IL11, IFN-γ, and HSP-70.

In yet other embodiments, a subject who is a candidate for treatment isidentified by the presence of one or more growth factors in the serum ofthe subject. These growth factors include, without limitation, FGF-2,G-CSF, GM-CSF, and M-CSF.

In some embodiments, a subject who is a candidate for treatment with thebiodiffusion chamber is identified by measuring the levels of a specificset of cytokines. In some embodiments, the subject has elevated levelsof these cytokines in comparison to a healthy subject. As used herein,the term “healthy subject” refers to a subject not suffering from canceror any other disease and not in need of treatment with the biodiffusionchamber.

In particular embodiments, the cytokines may be added to the chamber toaugment the anti-tumor immune response. For example, the cytokines addedto the chamber may be selected from the group consisting of CCL19,CCL20, CCL21, and CXCL12, and combinations thereof.

In certain embodiments, the circulating CD14+monocytes have an elevatedlevel of CD163 in comparison to a healthy subject. In some aspects, thelevels of CD163 on the circulating CD14+monocytes are elevated by atleast about 2 fold, at least about 3 fold, at least about 4 fold, atleast about 5 fold, at least about 10 fold, at least about 20 fold, atleast about 30 fold, at least about 40 fold, at least about 50 fold, atleast about 60 fold, at least about 70 fold, at least about 80 fold, atleast about 90 fold, or at least about 100 fold in comparison to ahealthy subject. In particular embodiments, the levels of CD163 on thecirculating CD14+ monocytes are elevated by about 2 fold in comparisonto a healthy subject.

In other embodiments, a subject who is a candidate for treatment hasserum that polarizes undifferentiated monocytes towards M2 cells. Incertain aspects, incubation of the subject's sera with undifferentiatedmonocytes induces the expression of one or more cell surface markers onthe monocytes including, but not limited to, CD11b, CD14, CD15, CD23,CD64, CD68, CD163, CD204, and/or CD206. In other aspects, incubation ofthe subject's sera with undifferentiated monocytes elevates theexpression of one or more cell surface markers on the monocytes incomparison to monocytes not incubated with the subject's sera. Incertain aspects, the cell surface markers include, but are not limitedto, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and/or CD206. Insome aspects, the levels of one or more surface markers are elevated byat least about 1.3 fold, at least about 1.5 fold, at least about 1.8fold, at least about 2 fold, at least about 3 fold, at least about 4fold, at least about 5 fold, at least about 10 fold, at least about 20fold, at least about 30 fold, at least about 40 fold, at least about 50fold, at least about 60 fold, at least about 70 fold, at least about 80fold, at least about 90 fold, or at least about 100 fold in comparisonto undifferentiated monocytes not incubated with the subject's sera. Inparticular embodiments, the levels of one or more surface markers areelevated by about 2 fold in comparison to undifferentiated monocytes notincubated with the subject's sera. Monocytes polarized by a subject'ssera may be measured using FACS.

Target Cells

Without being bound by theory it is thought that the AS ODN reduces thesubjects M2 cells and/or inhibits polarization of cells into M2 cells bydownregulating IGF-1R expression. In some embodiments, IGF-1R expressionin M2 cells is downregulated by at least about 1%, at least about 2%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 95% in comparison to cells not treated with the antisense. IGF-1Rexpression in M2 cells may be measured by quantitative RT-PCR.

In some embodiments, IGF-1R expression in M2 cells remains downregulatedin the subject for at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, or atleast about 6 weeks after receiving one dose of the antisense.

In some aspects, the downregulation of expression of IGF-1R in M2 cellscauses a selective reduction of M2 cells in a subject in comparison tocells not expressing IGF-1R. In certain embodiments, M2 cells in asubject are reduced by at least about 2%, at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% in comparison to asubject not treated with the antisense. In other embodiments, the M2cell population is eliminated. For example, after implantation of thebiodiffusion chamber, the M2 cell population may be about 1%, about 2%,about 5%, or about 10% of the population before implantation of thebiodiffusion chamber. M2 cells in a subject may be measured using FACS.In certain aspects, after treatment the M2 cells are eliminated; i.e.,undetectable by FACS. In other aspects, the decrease in M2 cells may bemeasured using a proxy assay; for example, serum from the subject may beobtained before and after treatment to assess its ability to polarize M2cells. Following treatment with methods disclosed herein, the ability ofthe serum to polarize M2 cells is reduced by about 80% to about 100%,about 20% to about 60%, or about 10% to about 50%.

In some embodiments, targeting the expression of IGF-1R in M2 cellscauses the M2 cells to undergo cell death. In certain embodiments, thecell death is necrosis. In other embodiments, the cell death isapoptosis. Apoptosis, for purposes of this disclosure, is defined asprogrammed cell death and includes, but is not limited to, regression ofprimary and metastatic tumors. Apoptosis is a programmed cell deathwhich is a widespread phenomenon that plays a crucial role in the myriadof physiological and pathological processes. Necrosis, in contrast, isan accidental cell death which is the cell's response to a variety ofharmful conditions and toxic substances. In yet other embodiments,targeting the expression of IGF-1R in M2 cells causes the M2 cells toundergo cell cycle arrest.

Kits

Preparation of a completed chamber requires multiple components andmultiple steps. In another aspects of the disclosure kits containingcomponents for practicing the methods disclosed herein are provided. Incertain aspects, the kits comprise the chamber body, which may bepresent in one portion or in two halves. Items to seal the chamber mayalso be included including one or more membranes, glues and solvents(e.g., an alcohol, or 2 dichloroethane). Optionally, the membrane may bysonically welded onto the chamber to create a seal. The kits include theantisense ODN. Optionally, the ODN may be divided into two portions. Afirst portion to treat the cells after surgical removal from thesubject, and a second portion to combine with the cells when introducedinto the subject. Other optional kit items include media for culturingthe cells, and antibiotics for preventing bacterial growth in the media.

Optionally, chambers in the kit may be pre-connected (e.g by suture) toeach other using an eyelet or other device attached to the chamber andadapted to receive the connecting material. Advantageously, bypre-connecting multiple chambers, the desired number of chambers may bereadily introduced and removed by the surgeon.

EXAMPLES Example 1

Vaccination with Autologous Tumor Cells and IGF-1R AS ODN in Patientswith Recurrent Glioblastoma

Criteria and Study Objective

Twelve subjects were enrolled for treatment after failure from standardtherapy. Each patient met the following criteria: age>18, a Karnofskyperformance score of 60 or better, and no co-morbidities that wouldpreclude elective surgical re-resection. The subjects were treated by 24hour implantation in the rectus sheath of ten biodiffusion chamberscontaining irradiated autologous tumor cells and IGF-1R AS ODN with theobjective of stimulating tumor immunity. Patients were monitored forsafety, clinical and radiographic as well as immune responses. Studyobjectives included assessment of safety and radiographic responses aswell as exploratory objectives looking at immune function and response.

TABLE 1 Summary of Patients enrolled Time Original LymphocyteIDH-1&IDH-2 between lymphocyte count at mutation/ surgeries Chamberscount enrollment Previous MGMT Subject Age KPS (weeks) (No.) (cells/mm2) (cells/mm 2) treatment methylation TJ01 39 70 177 10 N/A 400 S, RT +−/NA TMZ, Bev TJ02 57 80 90 9 N/A 1570 S, RT + −/methylated TMZ TJ03 7570 32 7 700 300 S, RT + −/NA TMZ TJ06/R¹ 66 80 54 8 2000 1300 S, RT +−/NA TMZ TJ07 43 80 215 10 500 430 S, RT + /methylated TMZ, Bev; RTOG0525 TJ08 55 80 52 8 1000 500 S, RT + −/TNS TMZ TJ09 57 80 61 7 1400 300S, RT + −/unmethylated TMZ, RTOG 0929 TJ10 47 60 376 7 N/A 1800 S, RT +−/methylated TMZ, Bev TJ11 39 70 32 11* 2400 200 S, RT + −/TNS TMZ TJ1260 80 74 7 1100 600 S, RT + −/TNS TMZ, Panobinostat TJ13 64 80 182 11N/A 2100 S, RT + −/methylated TMZ TJ14/R 77 90 30 9/11 1800 1100 S, RT +− TMZ unmethylated ¹Compassionate retreatment; *Protocol amendment toinclude control chamber filled with phosphate buffered saline; S:surgery; RT: radiation therapy; TMZ: temozolamide chemotherapy; Bev:bevacizumab chemotherapy; IDH-1: isocitrate dehydrogenase-1; NA: notavailable; TNS: tissue not sufficient

Experimental Protocol

Tumor tissue was surgically removed from patients using a tissueaspirator (NICO Myriad®) and placed into sterile tissue traps. Thesterile tissue traps were transferred to a designated BSL-2 facility,where the tumor tissue was processed and placed into biodiffusionchambers. The biodiffusion chambers were irradiated prior toimplantation.

The day following surgery to remove tumor tissue, ten irradiatedbiodiffusion chambers were implanted into the rectus sheath of thesubjects. After 24 hours, they were removed.

The biodiffusion chambers contained autologous tumor cells removed atsurgery. Prior to being added to the biodiffusion chambers, the cellswere pretreated overnight (approx. 12-18 hours) with a first amount (4mg/ml) of an 18-mer IGF-1R AS ODN with the sequence5′-TCCTCCGGAGCCAGACTT-3′ (NOBEL). Based on data showing that the AS ODNhas immunomodulatory properties, a second amount (2 μg) of exogenousNOBEL antisense was added to the chambers (C-v), and the chambers weresubsequently irradiated. Ten chambers were implanted in each patient. Aneleventh control chamber containing PBS (C-p) was also implanted.

Radiological Assessments

Serial imaging assessments were performed on Philips 1.5T and 3T MRIscanners and a GE 1.5T MRI scanner. Routine anatomic MRI features wererated by two neuroradiologists in all 12 patients. Physiologic MRItechniques of dynamic susceptibility weighted (DSC) MR perfusion and15-direction diffusion tensor imaging (DTI) were also utilized. MRperfusion and DTI post processing was performed on Nordic Iceworkstation (v.2.3.14). rCBV was calculated in relation to contralateralnormal white matter. Averaged diffusion coefficient (mean diffusivity)was calculated from the DTI data.

Immunological Assessments

Plasma leukopheresis was performed one week before surgery for baselineassessment of immune function. Blood was obtained post-operatively ondays 7, 14, 28, 42, 56, and every 3 months after vaccination. Sera andcell fractions were separated by centrifugation and cells were treatedwith red blood cell lysis buffer. White blood cells were eitherquantified by flow cytometry or stored in DMSO at −80° C. Serum sampleswere also stored at −80° C. Flow cytometry was performed using anEasyCyte 8HT (Millipore) and fluorescently-conjugated mAb specific forhuman CD4, CD8, CD11b, CD14, CD16, CD20, CD45, CD56, CD80, CD83, and CD86 (all from BD Biosciences), and CD163 (R&D Systems). Post-collectionanalysis was performed with FlowJo software (Tree Star Inc, Ashland,Oreg.). Serum cytokine factors were quantified using Luminex bead arrays(human cytokine/chemokine panels I, II, and III from Millipore) andHCMBMAG/MILLIPLEX Mag Cancer multiplex assay (emdmillipore.com). Thisincluded 6 serum markers for glioma related to stem cell functionincluding DKK-1, NSE, Osteonectin, Periostin, YKL-40, and TWEAK. Serumnitrate levels were assayed according to the Greiss method (Green L. C.,et al., 1982, Anal Biochem 126:131-8). T cell stimulation was performedwith phorbol 12-myristate, 13 acetate (PMA) and ionomycin as previouslydescribed (Verbrugge, I., et al., 2012, Cancer Res, 72:3163-74).

Cytokine/chemokine levels in tumor cell supernatant (SN) and explantedchamber contents were analyzed by Luminex kits as designated above.Membranes from paired vaccine and control chambers were embedded inparaffin for standard immunohistopathologic examination.

Tumor tissue sections were assessed by immunohistochemistry for GFAP(glial fibrillary acidic protein), IGF-1R, CD163, CD14, VWF (VonWillebrand Factor), CD4, and CD8 or fluorescence immunohistochemistryadapting the method described in Emoto, K., et al., 2005, HistochemCytochem 53:1311-21). Immunopositive cells were counted quantitativelywith Aperio or qualitatively by an experienced neuropathologist (LCK)using an ordinal scale from 0 (no staining) to 6 (strong diffusestaining) with staining intensity rated as low, moderate and strong andstaining patterns described as focal or diffuse. Post-mortem autopsy waslimited to examination of the brain and findings were compared toarchival paraffin blocks of previously treated or untreatedglioblastomas diagnosed at autopsy. Both canonical in vitro polarizationof naïve monocytes or mixing experiments involving naïve monocytesco-incubated with serum derived from trial subjects at enrollment wereperformed as previously described (Harshyne, L A, et al., 2015, NeuroOncol 18(2):206-15; Solinas, G., et al., 2010, J Immunol 185:642-52).

Statistical Analysis

The level of statistical significance between quantitative measures indifferent samples was determined by a two-tailed unpaired t-test ormatched pairs t-test with p<0.05. Survival analysis was performed byKaplan-Meier analysis and significance established by log rankcomparisons. All statistical analysis including mixture discriminantanalysis was performed with JMP v. 11 software (SAS, North Carolina).

Safety Assessment and Clinical Course

Only one severe adverse event (SAE) was related to the protocol (femoralvein thrombosis after leukopheresis). Nine patients succumbed to tumorprogression while three patients died from other causes. Five autopsieswere performed.

Median overall survival from initial diagnosis was 91.4 weeks (FIG. 2a )which compared favorably to other recurrent glioma immunotherapy trials.Two significantly different protocol survival cohorts of 48.2 and 10weeks were identified as longer and shorter survival cohorts,respectively (FIG. 2b ). Excluding one outlier (Patient TJ03), wedocumented a significant correlation between protocol survival anddegree of lymphopenia at enrollment (FIG. 2c ). Comparison of CBC valuesat initial diagnosis and at protocol enrollment indicated that the meanlymphocyte count had dropped significantly (65%) after standard therapy(N=8, p=0.012, paired t-test).

Radiographic Responses

Routine MRI features were assessed and rated by two neuroradiologists(K.S.T. and A.E.F.). In the longer cohort, diminished size ofenhancement and FLAIR envelope at the primary tumor site were observed,along with slower progression. Examples of anatomic responses in bothcohorts is shown in FIGS. 3a and 3b . Physiologic MRI measurementsaugmented these anatomic observations. Sequential DSC MR perfusion wasperformed in 7 patients, including 3 longer-term survivors (PatientsTJ03, TJ06, and TJ09) who had a paradoxical increase in relativeCerebral Blood Volume (rCBV) while improving clinically; however, thiseffect was transient and there was a more sustained decrease in rCBV.Sequential 15 directions DTI data included two long-term survivors(Patients TJ03 and TJ06) who showed apparent diffusion co-efficient(ADC) values increasing in the affected hemisphere, reflecting loss oftumor cellularity associated with disease regression. We noted a highcorrelation between the paradoxical rCBV response and increasing ADC notseen in the short cohort (FIGS. 3c and 3d ). Corresponding levels ofserum nitrate in the longer cohort reflected the likelihood that aninflammatory response had been initiated (data not shown).

Examination of Explanted Chambers v. Pen-Operative Serum by SurvivalCohort

Explanted chambers were structurally intact with no viable cells. Outersurfaces of membranes from both C-p and C-v chambers were coated withCD15+ and CD163+ cells, but with dramatically increased numbers on C-vmembranes (FIG. 4a ).

For the entire study cohort, analysis of the chamber soluble contentsrevealed significant elevations of a number of growth factors andcytokines/chemokines over matched perioperative serum levels, many ofwhich are well-documented in the glioma tumor microenvironment (TME).Thirty two of 78 cytokines/chemokines tested were significantly elevatedover serum and matched pairs analysis revealed significant elevations ofcytokines as noted in Table 2 below.

TABLE 2 Chamber Values by Survival Cohort Cytokines/chemokines presentin explanted chambers cytokine longer short P value Fold VEGF 4382 1207.001 3.63 PDGF-AA 272 90 .02 3.02 IL-11 1096 181 .002 6.06 CCL7/MCP-33293 1581 .001 2.08 CCL5/ 1484 169 .003 8.78 RANTES CCL22/MDC 385 1847.02 0.208 I-309 4.47 11.5 .02 0.389 MIP-1d 311 182 .002 1.71 Chambers:Matched pairs (chambers v. serum) cytokine longer P value short P valueVEGF 4028/81  .02 1178/80  .24 PDGF-AA  236/1803 .06  80/1431 .0011IL-11 958/54  .11 181/25  .047 CCL7/MCP-3 3276/29  .005 1581/18  .0004CCL5/ 1194/5571 .004  175/6201 <.001 RANTES CCL22/MDC 412/366 .81847/348  .09 MIP-1d 294/523 .02 182/473 .0095 Cancer markers present inexplanted chambers marker longer short P value Fold DKK1 629 1389 .020.452 NSE 7304 11712 .03 0.624 osteonectin 1022 1729 .02 0.591 periostin286 224 .10 1.27 TRAP5 1421 2441 .007 0.582 OPG 481 1409 .002 0.341YKL40 8615 12889 .13 0.668 TWEAK 190 171 .87 1.11 Chambers: Matchedpairs (chambers v. serum) Marker Longer P value short P value DKK1214/539 .16  457/1389 .01 NSE 6263/2182 .30 11723/1902  .01 osteonectin886/953 .70 1729/956  .04 periostin 249/532 .03 224/421 .004 TRAP5 998/1915 .13 2442/1940 .15 OPG 419/205 .48 1409/168  .01 YKL40 7300/12559 .19 12191/5574  .03 TWEAK 142/310 .15 324/217 .53

These elevations were interpreted as either cytokines/chemokinesproduced by the encapsulated tumor cells or factors produced by thelocal innate immune response that had diffused into the chambers.

Analysis of factors in chambers between survival cohorts revealedsignificant chamber elevations of VEGF, PDGF-α, IL-11, CCL5, MCP-3, andMIP-1 d in the longer cohort while a number of soluble cancer markerswere significantly elevated in the short cohort including NSE,osteonectin, and YKL40. Mixture discriminant analysis independentlyidentified these cohort differences (FIG. 4b ).

For both cohorts, both Periostin and CCL2 levels were significantlylower in the chambers (C-v) than serum or SN values, suggestingelimination of cells producing these chemokines in the chambers (FIG. 4c).

Serum Cytokines/Chemokines and PBMC after Vaccination by Survival Cohort

Levels of 24 of the 78 cytokines/chemokines assessed were significantlyhigher in serum from the longer cohort compared to the short cohort, asshown in Table 3 below.

TABLE 3 Serum Values by Cohort Cytokine/chemokine serum values (longcohort v. short cohort) cytokine long short P value CCL21 279 148 .0007CTACK 1313 1009 .01 Flt-3L 28 10.6 .02 Fractalkine 102 73 .004 I-30910.2 6.7 .02 IL-1RA 59 36 .001 IL-10 15 5 .003 IL-12-p40 41 3 .001 IL-138.6 3.1 .005 IL-15 11.4 4.6 .0005 IL-1a 41 3.6 .003 IL-1b 6.04 2.46 .003IL-2 7.18 2.87 .006 IL-3 5.06 3.13 .007 IL-5 2.8 1.35 <.0001 IL-9 5.613.07 .005 MCP-3 28 17 .002 MIP-3b 826 304 .005 MIP-1b 33 24 .01 SCF 11.83.6 .007 CXCL12 516 3.6 .008 TGF-α 2.91 1.93 .005 TNF-α 10.8 5.9 <.0001TPO 110 55 .02

A spike in serum CCL2 occurred after surgery but was absent atre-operation in two patients. CCL2 levels remained significantly higherthroughout the post-operative period in the short cohort. Thesepost-operative spikes were highly correlated with TNF-α spikes (FIGS. 5and 6).

Actual CD4 and CD8 T cell counts as well as dendritic cell (DC) countswere significantly higher in the longer cohort and perioperativeCD14+16-counts were significantly lower compared to the short cohort.There was a significant correlation between CD4 and DC cells and betweenCD4 and CXCL12 only in the longer cohort. Day 14 PBMC from the longersurvival subjects manifested significantly higher Th-1 cytokineproduction including IFNγ after stimulation with PMA and ionomycin thanthe short cohort (data not shown). Coordinated changes betweencirculating levels of T cells, monocytes, and pro-inflammatorychemokines/cytokines after vaccination were seen in three of foursubjects. The highest correlation was noted between total monocyte countand CD14+16-monocyte levels (FIGS. 5b and 5d ). An inverse relationshipbetween circulating T cell and monocyte numbers was also noted in thelonger cohort (FIG. 5) without significant differences in the shortcohort (FIG. 6). Predictable and reciprocal relationships betweenimmunosuppressive and pro-inflammatory cell populations as well asmonocyte-chemokine relationships suggested more immune fitness in thelonger cohort.

Examination of Paraffin Sections

Paraffin sections from surgical interventions through autopsies wereavailable for analysis in four cases allowing us to look at thepost-vaccination TME. We compared trial autopsies to autopsies fromre-operated and untreated GBM patients (FIG. 7). Immunostains revealed asignificant decrease in IGF-1R positive cells after vaccination inmatched pairs that was corroborated by fluorescence immunohistochemistry(FIGS. 7a and 7g ). Qualitative comparisons to either recurrent oruntreated glioma autopsies revealed abundant CD163 TAMs and IGF-1R+cellsin both, diminishing any concern of cell loss as autopsy artifact (FIG.7g ).

CD163 TAMs peaked at recurrence in matched comparisons to both initialsurgery and autopsy (FIGS. 7c and 7g ). Patients TJ06 and TJ10 supportedthese trends with evaluable samples through all phases of treatment(FIGS. 7b and 7d ). In the case of TJ06, CD163 cells dropped after thesecond vaccination and persisted through autopsy. This decreasecorrelated inversely with rCBV and ADC values as well as serum nitratelevels all of which increased after each vaccine (see FIG. 7f ).

Exploring an association with peripheral monocytes, a strong correlationwas noted between peripheral CD163+ monocytes and CD163 TAMs (FIG. 7e )in the short cohort not seen in the longer cohort (FIG. 7f ).

We did not see the emergence of T cell populations in the TME aftervaccination in either cohort.

Coincubation of Subject Serum with Undifferentiated Monocytes

To explore the genesis of the circulating CD163+ monocytes in thepatients we first polarized naïve monocytes with canonical M1 and M2cytokines IFN-γ and IL-4, respectively. We observed upregulation ofIGF-1R with M2 polarization only (FIG. 8a ). The M2 polarized CD163+population was selectively knocked down when incubated with IGF-1R ASODN (FIG. 8b ).

Subsequently, we coincubated naïve monocytes with serum obtained fromall study subjects and documented the emergence of CD163+ cells thatco-expressed both IGF-1R and PDL-1 (FIG. 8c ). When treated with IGF-1RAS ODN, cells expressing IGF-1R, PD-L1 and CD163 were significantlyknocked down in a parallel and dose-dependent manner (FIG. 8c )summarized in FIG. 8 d.

Discussion

The revised autologous cell/chamber-based glioblastoma multiforme (GBM)vaccination trial did not raise any significant safety concerns.

We identified two significantly different survival cohorts withdifferent responses to this vaccine paradigm. See FIGS. 5 and 6. Longercohort subjects typically exhibited elevated levels of tumor-specificantibody isotypes and cytokines/chemokines commonly associated with Th1immunity including IgG1, IgG3, IL12, CXCL10, CXCL12, CCL7, CCL19, andCCL21 following surgery and vaccination. Elevated levels of thesecytokines/chemokines were not seen in the short cohort. Accordingly,levels of cytokines/chemokines commonly associated with Th1 immunity(e.g., IgG1, IgG3, IL12, CXCL10, CXCL12, CCL7, CCL19, CCL21) may beassessed following surgery/vaccination to predict survival and to informfurther treatment strategies. Of interest, CCL21 and CXCL12 synergizewith CpG adjuvants and enhance the migratory and T cell stimulatorycapacity of DCs in a vaccination paradigm. Also, noted elevations ofGM-CSF, IL-6, Flt-3L and SCF in the longer cohort could enhance DCproliferation, and may have contributed to the significant 76% increasein pDCs after vaccination. The significant elevations of CD4 cells aswell as correlations between CD4 cells, pDC, and the cytokine CXCL12also suggests the successful induction of T cell proliferationfacilitated by CXCL12 during immune synapse.

Patients in the short survival cohort were typically subjected to alonger course of treatment prior to vaccination, leading tolymphophenia. Accordingly, vaccination is most effective whenadministered to patients with normal lymphocyte levels, i.e.non-lymphopenic patients. The treatment-induced lymphopenia and thelower CD4:CD8 ratio could also be ascribed to temozolamide. (Standard ofcare included conformal radiation with concomitant temozolamide followedby maintenance temozolamide initiated at 6 weeks post-surgery). Aconsequence of longer overall survival would include chronic exposure totumor antigens and ongoing glioma inhibitory signals leading to T cellexhaustion. Similarly, monocytes/macrophages had an apparent lack ofresponsiveness with only modest fluctuations after vaccination but adistinct correlation between peripheral CD14+16-cells and TAMs. TAMshave been associated with CCL2 production and this correlation couldreflect a closed loop amplification promoting tumor growth. Supportingthis, elevated serum CCL2 levels found in the short cohort have beenassociated with the mesenchymal gene expression profile and a poorprognosis in glioma patients.

The explanted chambers provided a unique snapshot of the encapsulatedTME and its commerce with the initial immune response. Cytokineelevations in the longer cohort chambers collectively indicated that thevaccinations induced a Th1 response, and serum from this cohortcontained tumor-specific antibody isotypes associated with Th1 immunity.Mixture discriminant analysis established associations with IFN-γ,TNF-α, and IL12 production.

In contrast, the short cohort chambers had greater elevations of cancermarkers reflecting the emergence of glioma stem cell (GSC)-associatedresistance after standard therapy. One conspicuous exception wasperiostin levels that were dramatically lower in all chambers comparedto paired serum values. Tumor-promoting cell populations in gliomainclude TAMs and GSC, the former supporting the latter in the sameperivascular niche (Zhou, W., et al., 2015, Nat Cell Biol 17:170-82) andboth representing strategic targets for treatment. M2 macrophagesrecruited by GSC-secreted periostin play a critical role in tumor growthand their elimination would have therapeutic advantage. The reduction inperiostin levels in chambers containing treated tumor cells suggeststhat GSCs secreting this factor themselves are a target for IGF-1R ASODN.

Notably, despite pre-existing immunosuppression (due to prior treatmentaccording to the standard of care) we documented radiographic andclinical improvements supported by a pro-inflammatory response aftervaccination in 4 of 12 patients. These patients also had a significantsurvival advantage on protocol. Exploring this survival differencefurther we noted a higher level of immune fitness in the longer cohort.

IGF-1R reduction after vaccination was associated with longer protocolsurvival in some subjects. Without being bound to any particular theory,it is possible that the IGF-1R+ cell populations are knocked down as aconsequence of type 1 immune mechanisms promoted in these individuals bythe vaccination paradigm.

As we have shown in vitro, IGF-1R AS ODN inactivates the CD163⁺ cellscontained in the vaccine preparation, thereby eliminating theirimmunomodulatory factors and promoting type 1 immunity. Moreover, anyIGF-1R AS ODN that diffuses out of the vaccine chamber has a similareffect on M2 macrophages that it reaches. This represents a novelplatform in which cells expressing a variety of tumor-promoting ligandsand factors, including PDL-1, other immunomodulatory factors, angiogenicfactors, nutrient support, and tumor invasiveness, are targeted.

Differences in the radiographic observations between the longer andshort survival patient cohorts provide further support for the conceptthat the vaccination paradigm has an impact on the broader glioma TME.Higher rCBV values are typically associated with tumor progression, andMR perfusion had only transient increases in the longer cohort, afinding not previously described. ADC measurements differentiated tumorprogression (lower values) from what we interpreted as cell loss (highervalues). Since this vaccination paradigm is associated with loss of bothIGF-1R cell and CD163+ TAM populations, it is possible that rising ADCvalues are a reflection of this.

In summary, we have established the safety profile of an improvedcombination glioma vaccine product and have documented alterations inimmune parameters associated with clinical and radiographicimprovements. With the promise of knocking down specific monocyte cellpopulations that promote tumor growth (e.g. CD163+ cells that co-expressIGF-1R), this paradigm offers a treatment scheme that does not result inimmune compromise.

Summary of Results

There were no Grade 3 toxicities related to protocol treatment andoverall median survival from initial diagnosis was 91.4 weeks (FIG. 2a). Two protocol survival cohorts with median survivals of 48.2 (“long”)and 10 weeks (“short”) were identified (FIG. 2b ). Longer survivalsubjects had imaging findings including transient elevations in cerebralblood volume (rCBV) and sustained elevations of apparent diffusioncoefficient (ADC) values interpreted as transient hyperemia and cellloss. Vaccine therapy resulted in the sustained loss of tumor-promotingCD163+ M2 and IGF-1R+ cell populations from the tumor microenvironment(TME). In vitro experiments were performed to explore the origin ofCD163+ T cells, and these experiments confirmed that subjects' serumdifferentiated immature monocytes into CD163+ cells with upregulation ofboth IGF-1R and PDL-1. Subsequent incubation with IGF-1R AS ODN resultedin a dose-dependent knock down of this M2 population which hasimplications for the immunogenicity of the encapsulated TME (tumormicroenvironment) treated with IGF-1R AS-ODN in the vaccine chamber. Thevaccine paradigm was well-tolerated with a favorable median survival.

Example 2

Vaccination of Newly Diagnosed Subjects with Glioblastoma

We demonstrated biological effectiveness of the vaccine protocolinvolving an autologous cell vaccine delivered as part of a formulatedcombination product involving implanted biodiffusion chambers inpatients with recurrent malignant gliomas who had failed standardtreatment,

Example 2 describes responses to administering the vaccine to newlydiagnosed glioma patients, including implanting 20 chambers for 24 or 48hours and 10 chambers for 24 or 48 hours. In each case, 2 μg of NOBELwas added into the chamber prior to irradiation in each case. Whencompared to standard of care in the first interim analysis, there weresignificant improvements in both progression-free survival and overallsurvival (FIG. 9). This was most notably due to the performance of thehigher dose cohorts after vaccination. We first noted significantlyhigher peak and mean interferon-gamma levels after vaccination in thenewly diagnosed patients compared to patient treated at recurrence. Inthe trial enrolling newly diagnosed glioma patients, we noted strikingand significant increases in IFN-y with each vaccine dose escalationwhen measuring aggregate serum measurements. The higher interferon-gammalevels with longer implantation correlated roughly with the rate atwhich the antisense diffuses out of the biodiffusion chamber.

These data, summarized in FIG. 10, illustrate that the autologouschamber vaccine induces anti-tumor responses in newly diagnosedglioblastoma patients. We further noted that increased IFNγ levels mayrepresent patient responses to tumor antigens and, if so, be a predictorof anti-tumor immunity and improved outcomes. Finally, the more robustresponse obtained in newly diagnosed GBM patients versus recurrentpatients, illustrates the impact of the subject's immune system andsupports vaccination of patients as a first-line therapy.

Example 3 Fully Formulated Chambers Have Greater Adjuvanticity

The fully formulated chamber includes the autologous tumor cells andother cells included in the tumor microenvironment (TME) treated 6 hoursprior to implantation with 4 mg/ml of IGF-1R AS ODN. The treated TME isthen encapsulated with exogenous addition of at least 2 μg of IGF-1R ASODN and the chamber is then irradiated with 5 Gy of gamma-irradiation.

We increased the number of chambers, meaning that the dose of IGF-1R ASODN received by each patient increased compared to previous studies. Forexample, twice the number of chambers implanted resulted in twice theamount of the antisense implanted and capable of diffusing out of thechamber, meaning the dose of AS ODN was about 40 μg, split between 20chambers.

The antisense sequence, particularly its palindromic CpG motif, and thedirect mixture with glioma cells in situ effectively initiate anti-tumorimmunity. Notably, the sense sequence with the same palindromic CpGmotif, is ineffective in the vaccine paradigm. Additionally, theantisense sequence must be directly admixed with the tumor inoculum inorder to see a satisfactory response. The dose of the IGF-1R AS ODN thatwill inhibit M2 monocyte polarization is at least an order of magnitudelower than the dose necessary to down-regulate expression of IGF-1R.

In preclinical animal modeling we assessed the efficacy of variousantigen preparations in restimulating therapeutic IFN-y-producing CD4 Tcells from C57 B6 mice that had rejected syngeneic GL261 glioma cellsimplanted in their cerebral cortex after vaccination. CD4 T cells wereisolated from the spleens of these animals using conventional approachesand added to bone marrow-derived dendritic cells from antigen naïve micethat had been incubated with various GL261 antigen preparations.Antigens recovered from the soluble fraction of a fully formulatedvaccine chamber including autologous tumor cells, exogenous antisense,and irradiation elicited significantly greater numbers ofIFN-γ-producing CD4 T cells than incomplete formulations.

Analysis of chambers containing different GL261 preparations implantedinto the flanks of C57BL/6 mice for 24 hours also provides evidence thatthe fully formulated chamber is most immunogenic. While IGF-1R AS ODNand irradiation each alone cause elevations of cytokines above a PBScontrol, 16 of 32 cytokines were significantly elevated over all othervariables, including irradiation alone, when combined with IGF-1R ASODN. Among these, at least 11 cytokines are associated with aninflammatory response, including IL-10, IL-6 and TNF-α which arecommonly produced by a radiation-induced pro-inflammatory cytokinenetwork.

A proinflammatory response to the fully formulated chambers wasvalidated in our second Phase 1 human trial for patients with recurrentglioblastoma. We noted two distinctly different survival cohorts aftervaccination and established associations between immune fitness, aproinflammatory response after vaccination, and longer survival(unpublished observations). In particular we noted an elevated CD4:CD8ratio after vaccination in the longer cohort that we interpreted aslocal TLR9 DC activation directing CD4+ cells toward a Th1 phenotypeperhaps augmented further by the irradiation of tumor cells in thechamber.

Example 4 Fully Formulated Chamber in Naïve Mice

In naïve C57B6 mice, implantation of a fully formulated vaccine chamberwas significantly more effective at eliciting an initial immune responsethan partially formulated chambers. Mice were vaccinated in the flankwith one chamber for 24 hours. Chamber contents varied from no contents(PBS), partially formulated chambers (GL261 glioma cells alone, GL261with AS ODN, or GL261 and 5Gy of irradiation), and fully formulatedchambers (GL261, AS ODN, and irradiation).

As shown in FIG. 11, there was a greater production of pro-inflammatorycytokines in mice implanted with a fully formulated vaccine chambercompared to mice implanted with partially formulated vaccine chambers(i.e. vaccine chambers containing tumor cells but no antisensemolecules).

Example 5 Dose-Dependent Dendritic Cell Activation in Normal Samples byIGF-1R AS ODN

PBMC from two normal donor sources were used to assess dose-dependent DCactivation by NOBEL antisense as well as the sequence used previously(DWA, 18-mer two codons upstream from the NOBEL sequence and describedin Andrews et al. (2001) “Results of a pilot study involving the use ofan antisense oligodeoxynucleotidedirected against the insulin-likegrowth factor type I receptor in malignant astrocytomas.” J Clin Oncol19:2189-2200).

PBMC were incubated overnight with the antisense sequences, along withthe sense sequence to the NOBEL antisense, then analyzed by flowcytometry, gating for a CD123+, CD68+ activated DC population. As shownin FIG. 12, the NOBEL antisense yielded a dose-dependent DC activationthat was significantly different from unstimulated controls or NOBELsense sequence, and more effective than the DWA sequence. These dataillustrate that, even compared to other IGF-1 AS, the NOBEL sequence isespecially effective.

Example 6

In Vitro T Cell Response from Contents of Fully Formulated ChamberUtilizing T Cells Derived from Vaccinated Mice

We hypothesized that, given the small pore size of the diffusion chamber(100 nm) that exosomes were the likely source of tumor antigen diffusingthrough the chamber membrane during implantation. C57B6 mice vaccinatedwith a flank injection that included GL261 glioma cells and IGF-1R ASODN were fully protected against a subsequent brain intra-parenchymaltumor challenge. We assessed vaccinated, tumor therapeutic T cellimmunoreactivity derived from these mice to contents of the fullyformulated chamber with Elispot assays for IFNγ using the followingantigen sources: 1/ Centrifuged supernatants from chambers loaded withGL261 cells and IGF-1R AS ODN irradiated and implanted in the mouseflank for 24 hours; 2/ Centrifuged supernatants from similarly preparedchambers incubated in isotonic PBS medium overnight at 37° C.; 3/Exosomes prepared from GL261 cells. These antigen preparations wereadded to dendritic cells from tumor antigen naïve mice and then added toCD4 T cells isolated from the spleens of GL261 immune mice or incubatedovernight prior to addition to the T cells to allow antigen processingand presentation. Following 24 hour coculture of the T cells antigen anddendritic cells the number of IFNγ-producing CD4 T cells was quantifiedin an Elispot assay. Chamber contents were compared to GL261 exosomes atvarious dilutions. Elispot results revealed a robust IFN-γ response onlywith chamber contents retrieved from 24 hour PBS incubation assayed withantigen presentation. Neither implanted chambers nor control Elispotassays in which dendritic cells were included without preincubationyielded significant differences from exosomes. These data reveal thatantigens derived from the TME are not exosomal in nature, are mostabundantly produced in irradiated chambers containing the tumor cellsand IGF-1R AS ODN, that they are expended during implantation, and thatthey require antigen presentation by DCs. Results are summarized in FIG.13.

Example 7 Biphasic Dose Response to M2 Monocyte/Macrophage Polarization

To determine the optimal dose of NOBEL IGF-1R AS-ODN to inhibit M2polarization in vivo, C57BL/6 mice were injected in the flank with 10⁶GL261 cells. 20 days later, the mice were given a single 0.75 or 0.075mg dose of NOBEL IGF-1R AS-ODN intraperitoneally. The mice were thenfollowed for tumor development.

The dose titrations of the NOBEL antisense on M2 generation in vivoyielded a paradoxical biphasic response. While doses at either extremeof a dose-seeking titration resulted in M2 monocyte knockdown,intermediate doses actually stimulated M2 monocyte generation. In US2017/0056430, it was shown that a single dose of 4 mg is highlyeffective in similar experiments. In the instant experiment, a singledose of 0.075 mg was highly effective, whereas an intermediate dose of0.75 mg was unexpectedly less effective. (FIG. 17). Without beinglimited, held or bound to any particular theory or mechanism of action,we hypothesize that the biphasic effect may be a consequence of theimmunostimulatory attributes of the NOBEL sequence.

The effective dose for inhibition of monocyte polarization by AS ODN isconsiderably lower than the dose necessary to downregulate IGF-1Rtranslation according to Watson-Crick base-pairing rules. Notably, invitro doses equivalent to the 0.075 mg dose per mouse have no effect oncells that are already expressing IGF-1R. In vitro titration experimentswith human monocytes reveal a substantial difference in the capacity ofIGF-1R AS-ODN treatment to prevent polarization as opposed to impact thephenotype or function of polarized M2 monocytes.

As shown in FIG. 14, the lowest dose achieves the same efficacy as thehighest dose suggesting a complex dynamic between the NOBEL antisenseand M2 generation. Based on the monophasic response to DC activation,the ideal chamber dose would be the point of maximal DC activation.

Example 8 Dose Response Curve for Inhibition of Monocyte Polarization byNOBEL

We performed a NOBEL antisense titration to levels in the aggregaterange of concentrations that feasibly would diffuse locally out of theimplanted chambers.

As shown in the FIG. 15, allogeneic naïve monocytes from three normalPBMC collections were incubated overnight with six different seraobtained from patients with glioblastoma, in the presence or absence ofdifferent concentrations of IGF-1R specific AS-ODN (NOBEL). Each coloreddot represents serum from an individual glioblastoma patient. Expressionof markers including CD163 was assessed by flow cytometry. CD163expression levels are presented as the mean fluorescence index of cellsstained with fluorescent conjugated CD163 antibodies.

Each patient's sera caused differentiation of MO monocytes into M2 CD163phenotype with upregulation of both IGF-1R and PDL-1. MO cells culturedwithout patient sera (ctrl) maintained very low levels of CD163 whileovernight incubation in sera strongly induced expression of this M2marker (untreated). The addition of IGF-1R specific AS-ODN to theculture media inhibited MO-M2 polarization as indicated by the elevatedexpression of CD163 in a dose-dependent manner. We noted a downwardtrend starting at 100 pg and reaching a significant level of inhibitionat 1 μg. These data confirm that excess antisense diffusing out of thechamber can facilitate the initiation of a Th1 response in the initialstages of innate immunity.

Example 9

Prevention of the Appearance of Anti-Inflammatory M2 Monocytes in MiceImplanted with CL261 Glioma Cells

C57BL/6 mice implanted with GL261 glioma cells develop tumors inparallel with elevated numbers of circulating CD163 expressing M2monocytes. We hypothesized that the glioma cells produce factors thatcause monocyte recruitment and polarization to M2. These cells theninfiltrate tumor tissues where their products promote tumor progression.Systemic treatment with IGF-1R AS-ODN may prevent the appearance of M2cells and thereby inhibit tumor formation.

C57BL/6 mice were implanted in the flank with 10⁶ GL261 cells and givena single dose of 4 mg NOBEL IGF-1R AS-ODN intraperitoneally orintravenously 20 days later. 14 days later peripheral blood was obtainedfrom the animals and circulating monocytes assessed by flow cytometryfor the expression of CD163. FIG. 18 shows a histogram of cell numbersexpressing CD163 (right hand peak) where the red line representsimplanted mice treated with PBS vehicle and the blue line implanted micetreated with the AS-ODN. The data shows that CD163+cells significantlydecline. The appearance of cells expressing CD204 or CD206 was similarlyinhibited (data not shown). Peripheral blood from normal, non-implantedmice did not contain cells with high levels of CD163, CD204, or CD206(data not shown).

Example 10

Systemic IGF-1R AS-ODN Treatment of Mice Implanted in the Flank withGlioma Cells Prevents the Development of Tumors.

C57BL/6 mice were implanted in the flank with 10⁶ GL261 cells and givena single 4 mg dose of NOBEL IGF-1R AS-ODN intraperitoneally orintravenously 20 days later, prior to the appearance of circulatingCD163-positive monocytes. Another group of C57BL/6 mice were injectedwith PBS as a control. Both groups of mice were then followed for tumordevelopment. As shown in FIG. 19, tumor incidence between the treatedand untreated groups was significantly different (*=p<0.05) with theNOBEL-treated mice much more like to remain tumor-free.

Example 11 Systemic IGF-1R AS-ODN Inhibition of Flank Glioma TumorGrowth is Independent of Anti-Tumor Immunity.

Tbet is a T-cell associated transcription factor, and Tbet deficientmice lack the ability to mount anti-glioma immunity. To test whetherIGF-1R AS-ODN inhibition of flank glioma tumor growth is independent ofanti-tumor immunity, Tbet deficient mice on a C57BL/6 background wereimplanted in the flank with 10⁶ GL261 cells and given a single 4 mg doseof NOBEL IGF-1R AS-ODN intraperitoneally or intravenously 20 days later.The mice were then followed for tumor development.

As shown in FIG. 20, tumor incidence between mice treated with PBS andmice treated with NOBEL IGF-1R AS-ODN was significantly different(*=p<0.05) despite the inability of Tbet deficient mice to mounttherapeutic anti-glioma immunity.

Example 12

Targeting Nestin+Stem Cells in the Chamber with NOBEL

We have shown that Nestin+ stem cells can be knocked down in adose-dependent manner by the NOBEL antisense in vitro and further thatthese cells are eliminated from the TME after the autologous cellvaccine (trial 14379-101, unpublished observations). As stem cells thatare part of the glioma tumor microenvironment (TME), selectivelyknocking them out has clear therapeutic benefit. With a morphology thatsupports an embryonic radial glial cell, these cells by their design andlong processes could serve as scaffolding allowing for deployment ofglioma cells throughout the brain. Removing them along with CD163 TAMscould reverse the invasive nature of these tumors as well as tumorgrowth itself. As a targetable cell in the chamber, antigens from thesecells could be very immunogenic and tumor-specific since they would beembryonic in origin. Nestin is primarily expressed in neuralprogenitor/stem cells and is located in the cytoplasm as a type VIintermediate filament. It has also been identified as a surface proteinand a biomarker for glioma stem cells. It would therefore be possible tobead-select and enrich this population thereby increasing theproinflammatory titer of the chamber. See Jin et al., “Cell surfaceNestin is a biomarker for glioma stem cells,” Biochem Biophys ResCommun. 2013 Apr. 19;433(4):496-501

Example 13 The Impact of Irradiation on the Chamber Formulation

During preparation of the fully formulated chamber, autologous tumorcells (i.e. freshly resected tumor tissue) are plated in serum-freeculture, optionally treated with a first amount of an IGF-1R AS ODN, andlater treated with ex vivo irradiation (FIGS. 1f and 1g ). A secondamount of IGF-1R AS ODN is added to the chamber before irradiation.

Since the autologous vaccination includes irradiation of the combinationproduct prior to implantation at a site remote from the tumor and ourdata support an immune response with tumor regression, these datasupport a novel abscopal effect. Typically, abscopal effects areattributed to activation of anti-tumor immunity after in situ radiationof a targeted tumor, which leads to tumor regression at sites distantfrom the radiation. In this particular formulation, the addition ofexogenous antisense with a CpG motif to the chamber, and subsequenttreatment with gamma-irradiation has been shown to up-regulate genesengaged in the activation, proliferation, and survival of memoryT-cells. Such a formulation also prevents the activation of genesinvolved in the generation of Tregs and the induction of immunetolerance. Additionally, down-regulation of the IGF-1R radiosensitizescells which are overexpressing this surface receptor. Coincubation withIGF-1R AS ODN also promotes apoptosis of targeted tumor cells (only invivo) and tumor-associated M2 macrophages. Irradiation with 5 Gy leadsto death of all encapsulated cells and causes the release of endogenousdanger signals known as danger/damage-associated molecular patterns (orDAMPS) that augment the presentation of tumor antigens released fromdying tumor cells.

Example 14 The Explanted Chamber as a Means of IdentifyingPro-inflammatory Agents for Future Chamber Formulations

The explanted biodiffusion chamber, retrieved after its application as adepot antigen device, also serves as repository documenting the initialimmune response corroborated in a preclinical mouse model and in a humantrial. Characterization of the chamber contents, with an appropriate PBS(dummy) chamber control, provides insight into both the host immuneresponse (in-diffusion of cytokines/chemokines above the dummy control)as well as production of cytokines/chemokines/DAMPS by cells within thechamber (undetectable in the dummy chamber). Consistent presence of anarray of cytokines informs exogenous additions of these cytokines tofuture formulations. As examples, CCL21 and CXCL, are both elevated inthe vaccine chambers over PBS chambers, synergize with CpG adjuvants andenhance the migratory and T cell stimulatory capacity of DCs in avaccination paradigm. See FIGS. 16 and 17. The exogenous addition ofthese cytokines to the chamber formulation could enhance the initialTh-1 response.

Example 15 Optimum Ratio of Cells to IGF-1R AS ODN in Chamber Leads toHigher Cytokine Values

Patients were vaccinated with 20 chambers each containing irradiatedtumor cells and AS NOBEL ODN (2 μg) for 48 hours. In each case, patientsalso received mandatory Thomas Jefferson University Hospital (TJUH)Standard of Care (SOC) therapy. Patients were followed to determineprogression-free survival (P-FS) (i.e., those patients both alive andshowing no development of cancer or remission) and overall survival (OS)at certain time-points. FIG. 21a-c illustrate responses at certaintime-points. FIGS. 24-27 illustrate patient outcomes and compares thosepatients treated (“vaccinated”) vs. the historic standard of care(“SOC”). To determine the optimum ratio of cells to IGF-1R AS ODN in thechambers, we measured pro-inflammatory cytokine levels in patient serumafter vaccination and compared these cytokine levels to cell number oftumor tissue removed from each patient.

Initially, as shown in FIG. 21a -c, a significant dose-dependentincrease in pro-inflammatory cytokines was observed in patient serum.Overall levels of IFN-γ were elevated quite significantly for thehighest dose cohort. The levels of IL12 and TNFα were also elevated inthis cohort.

Each of the three cytokine values from days 14-42 for each patient werepooled and plotted against IFNγ, IL12 and TNFa mean values. Twopolynomial plots with similar degree fits of 4 and 5 revealed peakpro-inflammatory cytokine values (FIG. 21d-f ).

FIGS. 24a and 24b show Kaplan-Meier curves illustrating progression-freesurvival and overall survival in the intention to treat group as awhole. In vaccinated patients, over about 35% were alive and wereprogression-free at 20 months. In contrast, less than 10% of SoC-treatedpatients showed progression-free survival at 20 months. Overall survivalwas similarly much improved with about 40% of patients surviving beyond25 months, whereas SOC-treatment shows around 5% survival at thattime-point. FIG. 24 b.

FIGS. 25a and 25b shows survival data for patients with a median age of61.5 years and matched such that the female/male numbers are 12/18 inboth groups. Again, the data illustrate the significantly improvedsurvival at various timepoints.

During the trial, some patients withdrew from protocol and others diedfrom unrelated causes. FIGS. 26a and 26b illustrate survival data absentdata from patients from those withdrawn patients or where deaths werefrom other causes. Again, the vaccinated patients perform significantlybetter. Certain patients were unable to complete the standard of care.Data excluding those patients is shown in FIGS. 27a and 27b . These dataconfirm that the vaccination approach is effective when standard of careprotocol is not followed.

FIGS. 28a and 28b illustrate the dosing effect of the cell number onpatient response. IFN-γ levels correspond to subject response. HigherIFN-γ levels are associated with better patient immune response andhence anti-tumor response. Here, we optimized that response bydetermining the correct titration of cells. The peak response is aroundthe 20 mark, i.e., 20 million cells, divided among 20 chambers. Thus,peak response is around 1 million cells/chamber while an excellentresponse is obtained with around 15 to 25 million cells, each dividedand implanted in 20 chambers; i.e. a range of 750,000 cells to 1,250,000cells per chamber. These data demonstrate the efficacy of the optimizedvaccination protocol.

Example 16

Enhanced Anti-Tumor Response Mediated by Vaccination with CellPopulation Enriched for Nestin Expression

The production of antigens by IGF-1R-treated glioma cells in chamberswas tested ex-vivo using glioma-immune T cells isolated from C57BL/6mice immunized using the chamber paradigm and challenged intra-craniallywith congenic GL261 cells to detect the presence of antigen. Mice toserve as donors of immune T cells were immunized as follows: Fullyformulated chambers filled with GL261 cells and antisense were implantedfor 24 hours in the flank. Chambers with only cells and no antisensewere also implanted as controls for antisense activity. Mice were bledthroughout the experiment and sera was tested for antibody reactivity toGL261 cells (FIGS. 30c, 30d ). At 35 days post-chamber implantation, themice were challenged intra-cranially with GL261 cells stereotactically.Survival and clinical signs of disease for the separate groups of micewere monitored for at least 40 days post-challenge. Survival andclinical disease score are shown in FIGS. 30a and 30b , respectively.

CD4+ T cells were isolated from the spleens of immunized mice usingmagnetic beads. Naive dendritic cells (DC), which were used to presentthe antigens to immune CD4 T cells, were isolated from the bone marrowof autologous, non-immune C57BL/6 mice. The DC were pulsed by overnightculture with GL261 antigens recovered from GL261 cells cultured inchambers overnight under different conditions, thereby mirroring what ishappening with respect to antigen production when similar chambers areimplanted in a subject. The chambers contained either GL261 cells aloneor GL261 with 3 different doses of antisense in phosphate bufferedsaline (PBS). Antisense at the different doses was added to antigenpreparations from G261 cells cultured without antisense to determinewhether the antisense content or the effect of antisense in the chambersis responsible for optimal antigen production. IFNγ production, believedto be the key measure of anti-tumor cell immunity, was used to assessthe stimulatory effects of the various antigen preparations on T cellactivation, with the specific number of responding cells quantified bythe ELISPOT assay, as depicted in FIG. 29 a.

To stimulate production of the antigen, we followed the in-vivo clinicalchamber paradigm. Approximately 1 million ex-vivo GL261 tumor cells wereinjected into chambers alone or with indicated antisense concentrationsand incubated overnight in the chamber which was placed in PBS). Thefollowing day, chamber content was extracted and used to pulse naïvedendritic cells. Chamber content which was not treated overnight withantisense was added to the dendritic cells with the indicated amounts ofNOBEL. Dendritic cells were also left naïve for control. Following anovernight pulse with antigen, dendritic cells were collected andincubated overnight with T cells from immunized animals in a cellculture plate coated with an ELIPSPOT detection antibody for thecytokine IFNγ. After overnight incubation, the coated plate wasprocessed and developed to enumerate the number of IFNγ-producingT-cells which responded to each respective antigen.

As shown in FIG. 29b , tumor antigens were detected in materialsrecovered from chambers containing GL261 cells plus antisense but notmaterials from chambers cultured with cells alone, even if antisense wasadded to the material when the DC were pulsed. This shows that thepresence of antisense in chambers with the glioma cells is required toproduce immunostimulatory tumor antigen.

To test the impact of overnight treatment with antisense, we alsoincubated cells overnight with 4 mg of antisense prior to addition ofthe cells to chambers. GL261 cells were plated in petri dishes andtreated overnight with 4 mg NOBEL per 1 million cells or were leftuntreated. The cells were then collected and placed into chambers at 1million cells and 2 μg NOBEL per chamber. The chambers were thenincubated overnight in PBS and the content was extracted the followingday. Dendritic cells were then pulsed with the chamber content and IFNγsecretion was measured as described above.

As shown in FIG. 29c overnight treatment of GL261 cells with antisenseenhances the amount of antigen produced by these cells as detected by anincrease in the numbers of tumor-immune T cells producing IFNγ when DCwere pulsed with GL261 cells treated with 4 mg antisense overnight.

To determine if the glioma tumor cell subset that expresses nestin isassociated with enhanced immunogenicity, mice were immunized withchambers with or without IMV-001 (NOBEL) antisense containing GL261cells grown under conditions that resulted in higher versus lower levelsof the protein nestin. Long-term protection against the subsequentintracranial implantation of GL261 glioma cells (FIG. 30a, 30b ) as wellas the production of GL261 antibody (FIG. 30c, 30d ) by the mice wereassessed.

Chambers containing GL261 cells with high levels of nestin and antisenseinduced considerably better immune protection than chambers with similarcells without antisense or chambers with low-nestin GL261, regardless ofwhether or not antisense was included. Chambered GL261 cells expressinghigh levels of nestin were also superior at inducing GL261-specificantibody production in the mice than those containing low nestin levels.However, with respect to antibody production, the inclusion of antisensehad minimal impact.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method for preparing a biodiffusion chamber forimplantation into a subject having cancer, the method comprising: (a)encapsulating tumor cells obtained from the subject into thebiodiffusion chamber in the presence of an IGF-1R AS ODN, wherein theratio of tumor cells to IGF-1R AS ODN in the chamber is in a range fromabout 3.75×10⁵: 1 μg to about 6.25×10⁵: 1 μg; wherein the tumor cellsare obtained from the subject using a tissue morselator, and (b)irradiating the biodiffusion chamber.
 2. The method of claim 1, whereinthe tumor cells are dispersed before encapsulating the tumor cells inthe chamber.
 3. The method of claim 1, wherein the cells are not exposedto temperatures above body temperature during removal from the subject.4. The method of claim 1, wherein the cells are not exposed totemperatures above 37° C. during removal from the subject.
 5. The methodof claim 1, wherein the tissue morselator comprises a sterile trap. 6.The method of claim 1, wherein the tissue morselator comprises ahigh-speed reciprocating inner cannula within a stationary outercannula.
 7. The method of claim 5, wherein the outer cannula comprises aside aperture, and further wherein the tumor cells are drawn into theside aperture by electronically controlled variable suction.
 8. Themethod of claim 1, wherein the tumor cells are enriched for nestinexpression before they are placed into the biodiffusion chamber.
 9. Themethod of claim 1, wherein the tumor cells are enriched for adherentcells compared to the tumor cells obtained from the subject.
 10. Themethod of claim 9, wherein the tumor cells consist essentially ofadherent cells.
 11. The method of claim 1, wherein the cells are treatedwith IGF-1R AS ODN before encapsulation into the chamber.
 12. The methodof claim 11, wherein the IGF-1R AS ODN is present at about 2 mg to about6 mg per million cells.
 13. The method of claim 12, wherein the IGF-1RAS ODN is present at about 4 mg per million cells.
 14. The method ofclaim 11, wherein the treatment with IGF-1R AS ODN prior toencapsulation is for up to about 18 hours.
 15. The method of claim 11,wherein the treatment with IGF-1R AS ODN prior to encapsulation is forabout 12 hours to about 18 hours.
 16. The method of claim 1, wherein theIGF-1R AS ODN has the sequence of SEQ ID NO:1.
 17. The chamber of claim1, wherein the IGF-1R AS ODN in the chamber is present at about 2 μg.18. The method of claim 1, wherein the irradiated tumor cells arepresent in a range from about 750,000 to about 1,250,000 per chamber.19. The method of claim 18 wherein the irradiated tumor cells arepresent at about 1,000,000 per chamber.
 20. A method of treating asubject having cancer, comprising implanting two or more biodiffusionchambers according to claim 1 into the subject.
 21. The method of claim20, wherein about 10 to about 30 biodiffusion chambers are implantedinto the subject.
 22. The method of claim 21, wherein about 10 to about20 biodiffusion chambers are implanted into the subject.
 23. The methodof claim 22, wherein the biodiffusion chambers are implanted into thesubject for about 48 hours.
 24. The method of claim 20, wherein thecancer is a brain cancer.
 25. The method of claim 24, wherein the braincancer is selected from a grade II astrocytoma, a grade AIIIastrocytoma, a grade AIII-G astrocytoma, and a grade IV astrocytoma(glioblastoma multiforme).
 26. The method of claim 25, wherein the braincancer is a grade IV astrocytoma (glioblastoma multiforme).
 27. Themethod of claim 20, wherein the method is performed withoutchemotherapy, without radiation therapy, or without both.
 28. The methodof claim 20, comprising a second implantation of chambers subsequent tothe first implantation.
 29. The method of claim 28, wherein the secondimplantation uses tumor cells obtained from the subject at the same timeas those cells obtained from the first implantation.
 30. The method ofclaim 28, wherein the second implantation uses tumor cells obtained fromthe subject after the first treatment is complete and the tumor hasrecurred or not responded to the first treatment.